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Allele specific DNA marker for fusarium resistance gene FocBo1 in Brassica oleracea.

ABSTRACT: The fusarium yellows resistance (YR) gene FocBo1 was previously identified and the DNA markers were developed to assist the breeding of YR cultivars in Brassica oleracea. However, the further analysis revealed discrepancies between the phenotypes and the genotypes predicted by those DNA markers in cabbage commercial cultivars. Since this discrepancy seemed to be due to unknown susceptible alleles of focbo1, we sequenced the gene in 19 accessions to determine the sequence variations between alleles and found that there were two resistant FocBo1 alleles and six susceptible alleles in the investigated population. The newly designed PCR markers detected three mutations in the susceptible alleles that generate premature termination codons. These were shown to accurately distinguish resistant and susceptible alleles in more than 200 accessions of B. oleracea inbred lines and cultivars. The study revealed that the locus is represented by 37.2% resistant and 62.8% susceptible alleles within seventy-eight commercial cultivars. Structural analysis of the gene revealed that a part of the allelic variation comes from intragenic recombination between alleles. Our results enable a more precise prediction of the phenotype by marker assisted selection, promoting the production of YR cultivars in B. oleracea.

SUBMITTER: Sato M

PROVIDER: S-EPMC6711725 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Allele specific DNA marker for fusarium resistance gene FocBo1 in Brassica oleracea.

Sato Maho M Shimizu Motoki M Shea Daniel J DJ Hoque Mozammel M Kawanabe Takahiro T Miyaji Naomi N Fujimoto Ryo R Fukai Eigo E Okazaki Keiichi K

Breeding science 20190508 2

The fusarium yellows resistance (YR) gene FocBo1 was previously identified and the DNA markers were developed to assist the breeding of YR cultivars in Brassica oleracea. However, the further analysis revealed discrepancies between the phenotypes and the genotypes predicted by those DNA markers in cabbage commercial cultivars. Since this discrepancy seemed to be due to unknown susceptible alleles of focbo1, we sequenced the gene in 19 accessions to determine the sequence var ...[more]

PMID: 31481840

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Project description:BackgroundCabbage 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.ResultsWe 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.ConclusionsThis work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.

Project description:Fusarium wilt caused by Fusarium oxysporum f. sp. conglutinans (FOC) is a destructive disease of Brassica crops, which results in severe yield losses. There is little information available about the mechanism of disease resistance. To obtain an overview of the transcriptome profiles in roots of R4P1, a Brassica oleracea variety that is highly resistant to fusarium wilt, we compared the transcriptomes of samples inoculated with FOC and samples inoculated with distilled water. RNA-seq analysis generated more than 136 million 100-bp clean reads, which were assembled into 62,506 unigenes (mean size = 741 bp). Among them, 49,959 (79.92%) genes were identified based on sequence similarity searches, including SwissProt (29,050, 46.47%), Gene Ontology (GO) (33,767, 54.02%), Clusters of Orthologous Groups (KOG) (14,721, 23.55%) and Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) (12,974, 20.76%) searches; digital gene expression analysis revealed 885 differentially expressed genes (DEGs) between infected and control samples at 4, 12, 24 and 48 hours after inoculation. The DEGs were assigned to 31 KEGG pathways. Early defense systems, including the MAPK signaling pathway, calcium signaling and salicylic acid-mediated hypersensitive response (SA-mediated HR) were activated after pathogen infection. SA-dependent systemic acquired resistance (SAR), ethylene (ET)- and jasmonic (JA)-mediated pathways and the lignin biosynthesis pathway play important roles in plant resistance. We also analyzed the expression of defense-related genes, such as genes encoding pathogenesis-related (PR) proteins, UDP-glycosyltransferase (UDPG), pleiotropic drug resistance, ATP-binding cassette transporters (PDR-ABC transporters), myrosinase, transcription factors and kinases, which were differentially expressed. The results of this study may contribute to efforts to identify and clone candidate genes associated with disease resistance and to uncover the molecular mechanism underlying FOC resistance in cabbage.

Project description:BACKGROUND:The basic helix-loop-helix (bHLH) is the second largest gene family in the plant, some members play important roles in pistil development and response to drought, waterlogging, cold stress and salt stress. The bHLH gene family has been identified in many species, except for Brassica oleracea and B. napus thus far. This study aims to identify the bHLH family members in B. oleracea, B. rapa and B. napus, and elucidate the expression, duplication, phylogeny and evolution characters of them. RESULT:A total of 268 bHLH genes in B. oleracea, 440 genes in B. napus, and 251 genes in B. rapa, including 21 new bHLH members, have been identified. Subsequently, the analyses of the phylogenetic trees, conserved motifs and gene structures showed that the members in the same subfamily were highly conserved. Most Ka/Ks values of homologous gene were < 1, which indicated that these genes suffered from strong purifying selection for retention. The retention rates of BrabHLH and BolbHLH genes were 51.6 and 55.1%, respectively. The comparative expression patterns between B. rapa and B. napus showed that they had similar expression patterns in the root and contrasting patterns in the stems, leaves, and reproductive tissues. In addition, there were 41 and 30 differential expression bHLH genes under the treatments of ABA and JA, respectively, and the number of down regulation genes was significantly more than up regulation genes. CONCLUSION:In the present study, we identified and performed the comparative genomics analysis of bHLH gene family among B. oleracea, B. rapa and B. napus, and also investigated their diversity. The expression patterns between B. rapa and B. napus shows that they have the similar expression pattern in the root and opposite patterns in the stems, leaves, and reproduction tissues. Further analysis demonstrated that some bHLH gene members may play crucial roles under the abiotic and biotic stress conditions. This is the first to report on the bHLH gene family analysis in B. oleracea and B. napus, which can offer useful information on the functional analysis of the bHLH gene in plants.

Dataset Information

Allele specific DNA marker for fusarium resistance gene FocBo1 in Brassica oleracea.

Publications

Allele specific DNA marker for fusarium resistance gene <i>FocBo1</i> in <i>Brassica oleracea</i>.

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