WO2008101343A1 - Identification d'un gène candidat de résistance à la jambe noire (leptosphaeria maculans) lepr3 chez le colza (brassica napus) - Google Patents

Identification d'un gène candidat de résistance à la jambe noire (leptosphaeria maculans) lepr3 chez le colza (brassica napus) Download PDF

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WO2008101343A1
WO2008101343A1 PCT/CA2008/000335 CA2008000335W WO2008101343A1 WO 2008101343 A1 WO2008101343 A1 WO 2008101343A1 CA 2008000335 W CA2008000335 W CA 2008000335W WO 2008101343 A1 WO2008101343 A1 WO 2008101343A1
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gene
srap
blackleg
lepr3
plant
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PCT/CA2008/000335
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English (en)
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Genyi Li
Zining Wang
Peter B. E. Mcvetty
Yu Chen
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University Of Manitoba
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • a and B groups of blackleg isolates defined on the basis of their symptoms on B. napus (Johnson and Lewis 1994).
  • the A group isolates (virulent) have been recently subdivided into PG2, PG3, PG4 and PGT with respect to differential reactions on cotyledons of the S. napus cultivars, 'Westar', 'Quinta', 'Glacier' and 'Jet Neuf (Mengistu 1991 , Koch et al. 1991 , Kutcher et al. 1993, Williams and Fitt 1999, Fernando and Chen 2003, Chen and Fernando 2005).
  • the B group isolates (avirulent) have been classified into three genetically distinct subgroups: NA1 , NA2 and NA3 (Kochet al.
  • R-genes such as Pto (Pseudomonas syringae pv. Tomato) encoding a serine/threonine protein kinase (Martin et al. 1993), belong to one group.
  • Pto Pseudomonas syringae pv. Tomato
  • the Cf ⁇ Cladosporium fulvum gene family in tomato represents a group which encodes proteins with leucine rich repeats (LRR) (Jones et al. 1994; Banerjee 2001 ).
  • NBS nucleotide binding site
  • LRR domain LRR domain
  • Map-based cloning is commonly employed for the previously described disease resistance genes and the first R gene cloned with this method was the tomato Pto gene (Martin et al. 1993).
  • LepR3 gene in a B. napus cultivar 'Surpass 400', which is a gene introgression from B. rapa subsp. sylvetris (Crouch et al. 1994. Anon. 2001 ), was targeted through map-based gene cloning.
  • Sequence related amplified polymorphism SRAP was used to construct a fine map for the LepR3 gene and a physical map was constructed using the Arabidopsis genome sequence as a reference.
  • a method of conferring blackleg resistance to a plant comprising: introducing a nucleic acid molecule comprising a nucleic acid molecule encoding LepR3 (SEQ ID NO: 1-6) operably linked to a promoter functional in said plant; and growing said plant under conditions whereby LepR3 is expressed; characterized in that said plant is more resistant to blackleg infection compared to a control plant of similar type grown under similar conditions.
  • a gene- silencing construct comprising at least 20 consecutive nucleotides of the Brassica Lep3R (SEQ ID NO: 1-6) sequence, shown in Figure 2.
  • Blackleg is the most devastating disease in several major canola producing areas worldwide and the most effective means to control this disease is through the development of disease resistance cultivars.
  • This gene can be used to isolate other homologous blackleg resistance genes from canola and other Brassica species through homologous functional analysis.
  • gene pyramiding through marker assisted selection can be implemented to improve the disease resistance. Any markers developed with the gene sequences are the best molecular markers without recombination between marker and trait that can be used in breeding programs through MAS. Genetic engineering can be used to transform the susceptible cultivars with the R-genes described here.
  • flanking SRAP markers on the ultradense map were used to pinpoint the region that contains the gene of interest.
  • flanking SRAP markers on the ultradense map were used to pinpoint the region that contains the gene of interest.
  • a closely linked SRAP marker with a genetic distance of 0.3 cM was easily found when the SRAP markers on the ultradense map were used to conduct co-segregation analysis with the disease resistance gene LepR3.
  • the Arabidopsis genomic sequence should be beneficial for gene cloning in Brassica crops which are closely related species to Arabidopsis. Broad conserved syntenic regions at the genome level and the gene linearity between Arabidopsis and Brassicas allows the use of gene order in Arabidopsis to find candidate genes for controlling traits in Brassicas. Since the conserved sequences between Arabidopsis and Brassicas are mostly located in open reading frames, it is important for a marker system to detect these gene regions. As reported by Li and Quiros (2001 ), nearly half of SRAP markers are located inside genes.
  • Plant disease resistance genes share similar protein structure and domains and can be classified into different groups. After finding the markers that completely co-segregate with the disease resistance gene LepR3 among a population of 3900 individuals, a search for R-gene-like protein was carried out in a region flanking these completely linked markers. Among over 100 genes, there is only one gene At5g14210 in Arabidopsis that bears the R-gene structure and this gene is just located in the region that is used for finding the closely linked SNP markers. The results suggested strongly that the ortholog of At5g14210 in B. napus is the right candidate for the disease resistance gene LepR3.
  • At5g14210 encodes a receptor-like kinase that has the leucine-rich repeats (LRR) and transmembrane domains. This protein is similar to the proteins produced by two bacterial blight disease resistance gene loci Xa21 and Xa26 in rice (Song et al. 1995; Sun et al. 2004). As will be appreciated by one of skill in the art, the use of silencing constructs based on LepR3 will facilitate the analysis and identification of other R- genes.
  • LRR leucine-rich repeats
  • a gene- silencing construct comprising at least 20, at least 25, at least 50, at least 75, at least 100 or at least 200 consecutive nucleotides of Brassica Lep3R (SEQ ID NO: 1-6) sequence, shown in Figure 2.
  • the nucleotide sequence may be derived from the sense or anti-sense of Lep3R.
  • the construct is an RNAi construct, although as will be appreciated by one skilled in the art, other suitable silencing constructs known in the art may also be used.
  • Blackleg mainly causes problems in canola and rapeseed production. It may also cause disease in other Brassica crops.
  • a method of conferring blackleg resistance to a plant comprising: introducing a nucleic acid molecule comprising a nucleic acid molecule encoding LepR3 (SEQ ID NO: 1-6) operably linked to a promoter functional in said plant; and growing said plant under conditions whereby LepR3 is expressed; characterized in that said plant is more resistant to blackleg infection compared to a control plant of similar type grown under similar conditions.
  • LepR3 sequences (SEQ ID Nos. 1-6) are shown in Figure 2. Accordingly, a Lep3R sequence corresponds to or may be selected from the group consisting of SEQ ID Nos 1 -6.
  • control plant may be an untransformed, mock transformed, vector transformed or other similar control typically used in such experiments. It is also of note that the control does not necessarily need to be repeated each time. It is of note that commonly used Brassica promoters such as CaMV 35S may be used in the above-described constructs.
  • 210Ay442, 0127Fr382 and 1128BG275 on the map were found to co- segregate with the resistance gene LepR3.
  • the linked SRAP molecular markers were analyzed with 908 F 2 plants and 2992 F 3 plants with 52 recombinants found between the marker 210Ay442 and 12117Ar 269.
  • the SRAP marker 0127Fr382 was the closest molecular marker to the resistance gene, showing a genetic distance of 0.3 cM.
  • the linked SRAP molecular markers G278, 1217Ar269, 210Ay442, 0127Fr382 and 1128BG275 were sequenced. After BLAST analysis against the Arabidopsis database, the sequence of SRAP marker 1128BG275 was found to have a match to At5g18840 and that of G278 a match to At5g57354 in Arabidopsis, respectively. Unfortunately there was no solid hit in Arabidopsis for the sequences of the remaining three SRAP markers 1217Ar269, 0127Fr382 and 210Ay442.
  • the flanking sequence of 1217Ar269 matched with the Arabidopsis gene At5g57830. Since the corresponding genes to the SRAP marker sequences were located in two syntenic regions in Arabidopsis, chromosome walking was performed in both regions using the Arabidopsis genome sequence as a reference.
  • SNPs that were located in four Brassica homologs of At5g57035, At5g57345, At5g57670 and At5g57830 in Arabidopsis were developed and used to analyze the 52 recombinants mentioned previously, the genetic map for these four SNPs showed the same gene order in B.
  • Primers designed with the sequence of At5g13930 in Arabidopsis were used to screen a B. rapa BAC library and a BAC clone A48M23 was selected. PCR products amplified from the BAC clone A48M23 were obtained with the primers designed with the sequence of At5g13950 in Arabidopsis. The sequence of the PCR products was homologous to At5g13950 in Arabidopsis. The genes on this ⁇ . rapa BAC clone suggested the gene order in this syntenic region would be the same as that in Arabidopsis.
  • SNP13930, SNP14060, SNP14210 and SNP14220 were developed with Arabidopsis genes At5g13930, At5g14060, At5g14210 and At5g14220, respectively. All these four SNPs showed no recombinant among these 52 recombinants, indicating that they were very close to the resistance gene LepR3. After searching a region of about 1 ,060kb (At5g13290-At5g16000), At5g14210 is a leucine-rich repeat transmembrane protein kinase, which suggesting that the orthologous gene of At5g14210 is a good candidate for blackleg resistance gene LepR3 in 'Surpass 400'.
  • Pycnidial inoculum of the blackleg isolate 87-41 was prepared according to the method described by Mengistu et al. (1993). The modifications were as follows: The cotyledons with lesions were collected and washed three times in sterilized distilled water in a laminar hood. The cotyledons were then treated with 15% (VA/) bleach for 20 minutes with occasional agitation. After three 2-minute washes with sterilized water, the cotyledons were transferred to Petri dishes with V8 agar medium (250ml V8 juice, 0.5g CaCO3 and 15g granulated agar per litre). The dishes were placed in a temperature and light controlled growth chamber.
  • V8 agar medium 250ml V8 juice, 0.5g CaCO3 and 15g granulated agar per litre. The dishes were placed in a temperature and light controlled growth chamber.
  • the cotyledons were full of black pycnidia and sometimes pink pycnidiospores were released.
  • the spores were discharged by washing and scraping the agar surface with sterilized glass.
  • the blackleg inoculum concentration was adjusted with distilled water to 2*10 7 spores/ml from the stock solution.
  • Cotyledons were punctured with sharp pointed forceps. Ten ⁇ l of spore suspension was placed on each puncture. The plants were kept at room temperature with light overnight for recovery. The plants were then placed in a controlled growth chamber (14 hrs light at 24 0 C during day time and 2O 0 C at night). In about 12 days, disease symptoms were fully developed, and the disease severity was rated according to the classification of 0-9 (Delwiche, 1980). Disease severity ratings of 0 to 4 were classified as resistant while ratings of 5 to 9 were classified as susceptible. The cultivars 'Westar' and 'Supass400' and their F1 progeny were used as controls for every inoculation run.
  • SRAP was performed as described by Sun et al. (2007).
  • the 'LIZ' color was sued for the size standard, while the other four colors were used to label SRAP primers.
  • the ultradense genetic recombination map with 13551 SRAP markers that was constructed with 58 DH lines from a cross of 'Westar' and 'Zhongyou 821 ' (Sun et al.
  • SRAP PCR products were separated with sequencing gels. The gels were stained with a silver staining kit (Promega, Toronto). The target markers were identified by comparing the band patterns with the marker patterns that were produced with the ABI 3100 Genetic Analyzer. DNA was eluted as described in Molecular Cloning (Sambrook and Russell, 2001 ). The DNA was reamplified and compared with the original SRAP profile to confirm the right position by running the PCR products on an ABI 3100 Genetic Analyzer. The confirmed DNA products were sequenced with a BigDye Terminator v3.1 kit (ABI, Toronto).
  • BLAST analysis of the marker sequences was performed with the TAIR Arabidopsis database. Sequences of some SRAP markers were found to be homologous to Arabidopsis genes. For the SRAP marker sequences that did not match with any gene in Arabidopsis, genome walking was implemented to extend their flanking regions and the extended sequences eventually hit Arabidopsis genes. Chromosome walking was initiated with the Arabidopsis genes flanking the landed genes by the SRAP markers. The Arabidopsis gene coding sequences were used to design primers that were used to amplify the DNA of 'Westar' and 'Surpass 400'. After sequencing these PCR products, SNPs were developed. These primers were also used to screen a S.
  • rapa BAC library for selecting the BAC clones anchoring the homologs of these Arabidopsis genes.
  • the end sequences of BAC clones were also used to develop SNPs. These SNPs were in turn used to screen the mapping populations.
  • the direction of chromosome walking was set by co- segregation analysis of the SNPs and the plant phenotypes in the mapping population.
  • the gene with the least recombinants was used to find a candidate gene that shared any similar domain to known R genes such as the genes with NBS and LRR domains.
  • a BAC library was constructed with a S. rapa male sterile line, following the protocol available at http://www.genome.arizona.edu/agi/.
  • a B. rapa male sterile line was used and a BAC cloning vector pCCBI BAC was purchased from Epicentre (Madison, Wisconsin). Nuclei imbedded in plugs were incubated in lysis buffer (0.5 EDTA pH9.0-9.3, 1% Na Lauryl sarcosine, 1.0mg/ml Proteinase K at 50 0 C for 48 hours with gentle shaking. After washing with TE buffer, the plugs were digested with Hindlll restriction enzyme for 5 minutes at 37 0 C.
  • the reaction was stopped by adding 5 ⁇ l EDTA (pH8.0).
  • the digested plugs were loaded into 1% low melting point agarose gel and run in BioRad CHEF-II system (Bio-Rad, Toronto). After 20 hours of running, part of the gel was stained in ethidium bromide solution. The gel with the appropriate DNA size (100-300kb) was excised and loaded to fresh gel for a second separation. The DNA in the slices was electroeluted and dialyzed against 0.05mM Tris-HCI (pH9.0-9.3). After ligation, competent E. coli DH10B cells (Invitrogen, Toronto) were used for transformation with a Bio-rad Gene Pulser (Bio-RAD, Toronto).
  • DNA sequencing was done with ABI 3100 Genetic analyzer following the ABI California protocol.

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Abstract

La jambe noire est la maladie la plus destructrice dans plusieurs régions de production de colza du monde entier et les moyens les plus efficaces de lutter contre cette maladie consistent à mettre au point des cultivars résistants à la maladie. Afin d'exploiter les gènes de résistance à la maladie de la jambe noire de manière plus efficace, on a ciblé par clonage positionnel un gène dominant de résistance à la maladie LepR3 chez le 'Surpass 400'. On a découvert, en utilisant comme plate-forme une carte de recombinaison génétique ultradense préalablement établie comprenant plus de 13500 marqueurs de polymorphismes d'amplification liés à la sequence (SRAP), que plusieurs marqueurs SRAP de la carte étaient étroitement liés au gène LepR3. Après avoir séquencé ces marqueurs SRAP et effectué une analyse BLAST des séquences, on a identifié les régions synténiques correspondantes chez Arabidopsis. La marche sur chromosome effectuée en utilisant les régions synténiques comme référence a permis d'identifier un gène qui code une kinase de type récepteur comprenant des domaines transmembranaires et des domaines de répétitions riches en leucine (LRR), portant les marques de certains gènes de résistance (R) identifiés chez d'autres espèces végétales.
PCT/CA2008/000335 2007-02-21 2008-02-21 Identification d'un gène candidat de résistance à la jambe noire (leptosphaeria maculans) lepr3 chez le colza (brassica napus) WO2008101343A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2015038470A1 (fr) * 2013-09-10 2015-03-19 Dow Agrosciences Llc Marqueurs moléculaires du gène rlm4 de résistance au charbon symptomatique chez brassica napus et leurs procédés d'utilisation
WO2015038469A1 (fr) * 2013-09-10 2015-03-19 Dow Agrosciences Llc Marqueurs moléculaires du gène rlm2 de résistance au charbon symptomatique chez brassica napus et leurs procédés d'utilisation
CN105567844A (zh) * 2016-02-05 2016-05-11 杭州市农业科学研究院 鉴定猴头菇猴杰2号或大猴头的引物对及方法
US10767190B2 (en) 2015-12-15 2020-09-08 Basf Agricultural Solutions Seed, Us Llc Brassicaceae plants resistant to Plasmodiophora brassicae (clubroot)
WO2023004429A1 (fr) 2021-07-23 2023-01-26 BASF Agricultural Solutions Seed US LLC Plantes résistant à la jambe noire et procédés d'identification de plantes résistant à la jambe noire

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EP1547462A1 (fr) * 2003-12-24 2005-06-29 Bayer BioScience N.V. Plante Brassica résistante au champignon Leptosphaeria maculans (nécrose du collet du colza)

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WO2001018221A1 (fr) * 1999-09-08 2001-03-15 Plant Bioscience Limited Gene de resistance de plante
EP1547462A1 (fr) * 2003-12-24 2005-06-29 Bayer BioScience N.V. Plante Brassica résistante au champignon Leptosphaeria maculans (nécrose du collet du colza)

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WANG Y. ET AL.: "Constitutive expression of pea defense gene DRR206 confers resistance to blackleg (Leptosphaeria maculans) disease in transgenic canola (Brassica napus)", MOLECULAR PLANT-MICROBE INTERACTIONS, vol. 12, no. 5, 1999, pages 410 - 418, XP002158024 *
WRETBLAD S. ET AL.: "Overexpression of a Brassica nigra cDNA gives enhanced resistance to Leptosphaeria maculans in B. napus", MOLECULAR PLANT-MICROBE INTERACTIONS, vol. 16, no. 6, June 2003 (2003-06-01), pages 477 - 484 *
YU F. ET AL.: "Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris", GENOME, vol. 51, no. 1, January 2008 (2008-01-01), pages 64 - 72 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015038470A1 (fr) * 2013-09-10 2015-03-19 Dow Agrosciences Llc Marqueurs moléculaires du gène rlm4 de résistance au charbon symptomatique chez brassica napus et leurs procédés d'utilisation
WO2015038469A1 (fr) * 2013-09-10 2015-03-19 Dow Agrosciences Llc Marqueurs moléculaires du gène rlm2 de résistance au charbon symptomatique chez brassica napus et leurs procédés d'utilisation
AU2014318042B2 (en) * 2013-09-10 2017-08-10 Corteva Agriscience Llc Molecular markers for blackleg resistance gene Rlm4 in Brassica napus and methods of using the same
AU2014318041B2 (en) * 2013-09-10 2017-10-26 Corteva Agriscience Llc Molecular markers for blackleg resistance gene Rlm2 in Brassica napus and methods of using the same
US10767190B2 (en) 2015-12-15 2020-09-08 Basf Agricultural Solutions Seed, Us Llc Brassicaceae plants resistant to Plasmodiophora brassicae (clubroot)
CN105567844A (zh) * 2016-02-05 2016-05-11 杭州市农业科学研究院 鉴定猴头菇猴杰2号或大猴头的引物对及方法
CN105567844B (zh) * 2016-02-05 2018-12-11 杭州市农业科学研究院 鉴定猴头菇猴杰2号或大猴头的引物对及方法
WO2023004429A1 (fr) 2021-07-23 2023-01-26 BASF Agricultural Solutions Seed US LLC Plantes résistant à la jambe noire et procédés d'identification de plantes résistant à la jambe noire

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