EP4164372A1 - Aphis gossypii resistance in cultivated capsicum plants - Google Patents
Aphis gossypii resistance in cultivated capsicum plantsInfo
- Publication number
- EP4164372A1 EP4164372A1 EP21729330.7A EP21729330A EP4164372A1 EP 4164372 A1 EP4164372 A1 EP 4164372A1 EP 21729330 A EP21729330 A EP 21729330A EP 4164372 A1 EP4164372 A1 EP 4164372A1
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- European Patent Office
- Prior art keywords
- snp
- nucleotide
- seq
- plant
- genotype
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/021—Methods of breeding using interspecific crosses, i.e. interspecies crosses
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
- A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
- A01H1/127—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for insect resistance
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
- A01H6/822—Capsicum sp. [pepper]
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- 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
-
- 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
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to the field of pepper breeding.
- a Quantitative Trait Locus located on chromosome 11 of the cultivated pepper genome, which increases resistance (or tolerance) against the cotton aphid, Aphis gossypii.
- the QTL is herein referred to as QTL11. It was identified in a wild Capsicum accession, an accession of Capsicum haccatum var. haccatum having small, pungent fruits (referred to as the QTL11 donor) and introgressed into cultivated sweet pepper, Capsicum annuum.
- a representative sample of seeds (BC2F3) of a C. annuum backcross line (BC2F2) comprising QTL 11 from the wild Capsicum donor in homozygous form has been deposited and from the deposit (NCIMB 43605), or from descendants of this deposit, the QTL can be easily transferred into any other Capsicum plant, especially any other plant of the species Capsicum annuum, such as bell peppers, sweet peppers, cayenne peppers, paprika and jalapenos.
- Capsicum annuum Apart from the transfer into cultivated Capsicum annuum, it can also be transferred into other domesticated Capsicum species, especially into lines or varieties of Capsicum haccatum, Capsicum frutescens, Capsicum chinense or Capsicum pubescens.
- embryo rescue and/or bridge species can be used to transfer the QTL to such other species.
- wild Capsicum donors especially wild Capsicum haccatum accessions
- wild Capsicum haccatum accessions can be identified which comprise the same QTL, e.g. comprising the same SNP haplotypes for QTL 11 or the same SNP haplotype for at least several, e.g.
- SNP markers especially for at least 1, 2, 3, 4, 5, 6, 7, 8 or more consecutive SNP markers, of the SNP markers selected from SNP_01 to SNP_18, or from SNP_02 to SNP_17, or from SNP_02 to SNP_15, or from SNP_02 to SNP_12, or from SNP_03 to SNP_10, or SNP_02 to SNP_08, or especially from SNP_01 to SNP_09, or from SNP_02 to SNP_09, or from SNP_01 to SNP_08, or from SNP_02 to SNP_08, or from SNP_02 to SNP_08, or from SNP_01 to SNP_07, or from SNP_02 to SNP_07, or from SNP_01 to SNP_06, or from SNP_02 to SNP_06, or from SNP_01 to SNP_04 or from SNP_02 to SNP_04, or from SNP_02 to SNP_03, or from SNP_03 to SNP_04.
- the location of QTL11 has been fine-mapped to he in between SNP 01 (at nucleotide 101 of SEQ ID NO: 1) and SNP_09 (at nucleotide 101 of SEQ ID NO: 9), especially to lie in between SNP_01 or SNP_02 (at nucleotide 101 of SEQ ID NO: 2) and SNP_06 (at nucleotide 101 of SEQ ID NO: 6) or to he between SNP_01 or SNP_02 (at nucleotide 101 of SEQ ID NO: 2) and SNP_05 (at nucleotide 101 of SEQ ID NO: 5), especially between SNP_01 or SNP_02 and SNP_04 (at nucleotide 101 of SEQ ID NO: 4), with SNP_03 at nucleotide 101 of SEQ ID NO: 3 being the most closely linked marker.
- other wild Capsicum donors comprising QTL 11 can be identified by comprising the same donor SNP haplotype for one or more (e.g. at least 1, 2, 3, 4, 5, 6, 7 markers) or all markers for SNP_01 to SNP_09 or SNP_02 to SNP_09, or for SNP_01 to SNP_08 or for SNP_02 to SNP_08, or for SNP_01 or SNP_02 to SNP_07, or for SNP_01 or SNP_02 to SNP_06, or for SNP_01 or SNP_02 to SNP_05, or for SNP_01 or SNP_02 to SNP_04, SNP_01 or SNP_02 to SNP_03 or SNP_03 to SNP_04.
- SNP haplotype for one or more (e.g. at least 1, 2, 3, 4, 5, 6, 7 markers) or all markers for SNP_01 to SNP_09 or SNP_02 to SNP_09, or for SNP_01 to SNP_08 or for SNP_02 to
- wild Capsicum donors comprising QTL11 can be identified by comprising the same donor SNP haplotype for the SNP markers in the fine mapped regions, especially SNP_01 or SNP_02 to SNP_06 or for SNP_01 or SNP_02 to SNP_05, or for SNP_01 or SNP_02 to SNP_04, or for SNP_01 or SNP_02 to SNP_03, or for SNP_03 to SNP_04, or for at least 1, 2 or 3 SNP markers selected from SNP_02 to SNP_06.
- wild Capsicum donors comprise at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally one or more other donor SNP nucleotides as mentioned, especially at least the donor nucleotide for SNP_02 and/or SNP_04.
- a cultivated Capsicum plant of the species C. annuum, C. chinense, C. frutescens, C. baccatum or C. pubescens comprising an introgression fragment from a wild Capsicum baccatum accession, on chromosome 11
- said introgression fragment comprises Quantitative Trait Locus QTL 11 located in between SNP_01 at nucleotide 101 of SEQ ID NO: 01 and SNP_09 at nucleotide 101 of SEQ ID NO: 09, or in between SNP_02 at nucleotide 101 of SEQ ID NO: 02 and SNP_09 at nucleotide 101 of SEQ ID NO: 09, wherein said QTL 11 confers a reduced susceptibility to Aphis gossypii, and wherein the introgression fragment comprises a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3, and optionally the SNP haplotype for at least 2, 3, 4, 5, 6 or
- baccatum accession comprising a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally comprising the donor SNP haplotype for at least 2, 3, 4, 5, 6 or 7 of the SNP markers of SNP_02 to SNP_08, especially comprising a Thymine for SNP_02 at nucleotide 101 of SEQ ID NO: 2 and/or an Adenine for SNP_04 at nucleotide 101 of SEQ ID NO: 4.
- a cultivated Capsicum plant of the species C. annuum, C. chinense, C. frutescens, C. baccatum or C.
- said introgression fragment comprises Quantitative Trait Locus QTL11 located in between SNP_01 at nucleotide 101 of SEQ ID NO: 01 and SNP_09 at nucleotide 101 of SEQ ID NO: 09, or in between SNP_02 at nucleotide 101 of SEQ ID NO: 02 and SNP_09 at nucleotide 101 of SEQ ID NO: 09, wherein said QTL11 confers a reduced susceptibility to Aphis gossypii, and wherein the introgression fragment comprises a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3, and optionally further the SNP haplotype: a Thymine for SNP_02 at nucleotide 101 of SEQ ID NO: 2, and/or an Adenine for SNP_04 at nucleotide 101 of SEQ ID NO: 4, wherein the
- baccatum accession comprising a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 at nucleotide 101 of SEQ ID NO: 2 and/or an Adenine for SNP_04 at nucleotide 101 of SEQ ID NO: 4.
- a cultivated Capsicum plant of the species C. annuum, C. chinense, C. frutescens, C. baccatum or C. pubescens comprising an introgression fragment from a wild Capsicum baccatum accession, on chromosome 11
- said introgression fragment comprises Quantitative Trait Locus QTL 11 located in between SNP_01 at nucleotide 101 of SEQ ID NO: 01 and SNP_08 at nucleotide 101 of SEQ ID NO: 08 or in between SNP_01 and SNP_07 at nucleotide 101 of SEQ ID NO: 7, or in between SNP_02 at nucleotide 101 of SEQ ID NO: 02 and SNP_08 at nucleotide 101 of SEQ ID NO: 08 or in between SNP_02 and SNP_07, wherein said QTL 11 confers a reduced susceptibility to Aphis gossypii, and wherein the introgression fragment comprises a Cyto
- baccatum accession comprising a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally comprising the donor SNP haplotype for at least 2, 3, 4, 5, 6 or 7 of the SNP markers of SNP_02 to SNP_07 or of SNP_02 to SNP_08.
- a cultivated Capsicum plant of the species C. annuum, C. chinense, C. frutescens, C. baccatum or C. pubescens comprising an introgression fragment from a wild Capsicum baccatum accession, on chromosome 11
- said introgression fragment comprises Quantitative Trait Locus QTL 11 located in between SNP_02 at nucleotide 101 of SEQ ID NO: 02 and SNP_06 at nucleotide 101 of SEQ ID NO: 06, or in between SNP_01 and SNP_06, wherein said QTL11 confers a reduced susceptibility to Aphis gossypii
- the introgression fragment comprises a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3, and optionally the SNP haplotype for at least 2, 3, 4, or 5 of the SNP markers selected from: a Thymine for SNP_02 at nucleotide 101 of SEQ ID
- baccatum accession comprising a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally comprising the donor SNP haplotype for at least 2, 3, 4 or 5 of the SNP markers of SNP_02 to SNP_06.
- a cultivated Capsicum plant of the species C. annuum, C. chinense, C. frutescens, C. baccatum or C. pubescens comprising an introgression fragment from a wild Capsicum baccatum accession, on chromosome 11
- said introgression fragment comprises Quantitative Trait Locus QTL 11 located in between SNP_01 at nucleotide 101 of SEQ ID NO: 01 and SNP_05 at nucleotide 101 of SEQ ID NO: 05, or between SNP_02 and SNP_05, wherein said QTL 11 confers a reduced susceptibility to Aphis gossypii
- the introgression fragment comprises a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3, and optionally the SNP haplotype for at least 2, 3, 4, or 5 of the SNP markers selected from: a Thymine for SNP_02 at nucleotide 101 of SEQ ID
- baccatum accession comprising a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally comprising the donor SNP haplotype for at least 2, 3, 4, or 5 of the SNP markers of SNP_02 to SNP_05.
- a cultivated Capsicum plant of the species C. annuum, C. chinense, C. frutescens, C. baccatum or C. pubescens comprising an introgression fragment from a wild Capsicum baccatum accession, on chromosome 11
- said introgression fragment comprises Quantitative Trait Locus QTL 11 located in between SNP_02 at nucleotide 101 of SEQ ID NO: 02 and SNP_04 at nucleotide 101 of SEQ ID NO: 4, or between SNP_01 and SNP_04, wherein said QTL 11 confers a reduced susceptibility to Aphis gossypii
- the introgression fragment comprises a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3, and optionally: a Thymine for SNP_02 at nucleotide 101 of SEQ ID NO: 2 and/or an Adenine for SNP_04 at nucleotide 101
- baccatum accession comprising a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally comprising the donor SNP haplotype for SNP_02 and/or SNP_04, or for at least 2, 3, 4 or 5 of the SNP markers of SNP_02 to SNP_06.
- the QTL lies physically in the genomic region of chromosome 11 which region starts at the nucleotide of one of the flanking markers and ends at the nucleotide of the other flanking marker.
- the physical position of a SNP marker can also be indicated as the nucleotide on the reference genome, as e.g. shown in Table 2 or 3.
- Seeds comprising the introgression fragment (which comprises QTL11) from the wild donor, in homozygous form, and comprising donor SNP markers / sequences indicative of (linked to) the introgression fragment in homozygous form, were deposited by Nunhems B.V. under accession number NCIMB 43605.
- the seeds comprise the donor SNP markers for all of SNP_01 to SNP_18 in homozygous form. See also Table 1. Aphids are damaging pests in pepper cultivation worldwide. Several aphids can cause problems in pepper cultivation, including Myzus persicae, Myzus nicotianae, Aulacorthum solani, Macrosiphum euphorbiae and Aphis gossypii.
- C. baccatum accession PB2013071 In pepper a resistance QTL against Myzus persicae (Green Peach Aphid) was identified on chromosome 2 of C. baccatum accession PB2013071 (Sun et al. 2020, Theor Appl Genet 133, 227-237). C. baccatum accession PB2013071 had previously been identified as one of three C. baccatum accessions which were shown to be resistant or intermediately resistant to Myzus persicae by negatively affecting aphid survival and reproduction. Accession PB2013071 showed the highest level of resistance, which severely impaired phloem uptake by the aphid and induced callose deposition in the sieve elements during aphid feeding (Sun et al. 2018, BMC Plant Biol 18:138).
- a cultivated pepper plant comprising an introgression fragment on chromosomes 11
- the introgression fragment significantly increases resistance of the cultivated pepper plant comprising the introgression compared to the control pepper plant (e.g. genetic control line) lacking the introgression.
- one or more molecular markers especially Single Nucleotide Polymorphisms or SNPs and/or the sequences comprising the SNPs which are present on the introgression fragment and are linked to QTL 11 and which are indicative of the presence of the introgression fragment (and of QTL 11) and methods of using such markers are provided herein for QTL11.
- seeds, plant parts, cells and/or tissues comprising QTL11 on chromosome 11 are provided.
- Chromosome 11 has a size of around 118.8 cM in the integrated genetic map of pepper, meaning that the region in which QTL 11 has been mapped (and in which SNP_01 to SNP_18 are found) is about 1/6* of chromosome 11.
- SNP markers SNP_01 to SNP_18 are linked to QTL 11, with SNP_06, SNP_07 and SNP_08 being the most significant markers in the initial mapping.
- the narrowed down region is about 1/10 th of the entire chromosome or less. Further fine-mapping can be done to further narrow down the region.
- the SNP haplotype of the wild donor is present in the deposited seeds for all of SNP_01 to SNP_18 in homozygous form.
- the donor SNP haplotype for at least at least 1, 2, 3, 4, 5, 6, 7, 8 or more SNP markers of SNP_01 to SNP_18, or SNP_01 (or SNP_02) to SNP_09 or preferably SNP_02 to SNP_06 or SNP_02 to SNP_05 or SNP_02 to SNP_04, especially for at least 1, 2, 3, 4, 5, 6, 7, 8 or more consecutive SNP markers, of the SNP markers selected from SNP_01 to SNP_18, or SNP_01 (or SNP_02) to SNP_09 or preferably SNP_02 to SNP_08, or SNP_02 to SNP_07, SNP_02 to SNP_06, or SNP_02 to SNP_05, or SNP_02 to SNP_04, or from SNP_02 to SNP_17, or from SNP_02 to SNP
- cultivated Capsicum plants (of a domesticated Capsicum species, especially C. annuum ), seeds, plant parts, cells and/or tissues comprising an introgression fragment from a wild Capsicum, especially from a wild C. baccatum donor are provided, whereby the introgression fragment comprises QTL11 or a functional variant of QTL11.
- cultivated Capsicum plants, seeds, plant parts, cells and/or tissues comprising a sub-fragment of an introgression fragment from a wild Capsicum donor, especially from a wild C. baccatum donor accession are provided, whereby the sub-fragment comprising QTL11 or a functional variant of QTL11.
- QTL11 is in one aspect located in the region starting at SNP 01 at nucleotide 101 in SEQ ID NO: 1 and ending at SNP_18 at nucleotide 101 in SEQ ID NO: 18.
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_02, SNP_03, SNP_04, SNP_05, SNP_06, SNP_07 and/or SNP_08, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the (introgression fragment comprising) QTL11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SNP markers selected from SNP_01 to SNP_18, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive markers selected from SNP_01 to SNP_18, preferably including SNP_03 and optionally SNP_02 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 and/or an Adenine for SNP_04.
- QTL 11 is in one aspect located in the region starting at SNP_02 at nucleotide 101 in SEQ ID NO: 2 and ending at SNP_17 at nucleotide 101 in SEQ ID NO: 17.
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_02, SNP_03, SNP_04, SNP_05, SNP_06, SNP_07 and/or SNP_08, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the (introgression fragment comprising) QTL11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SNP markers selected from SNP_02 to SNP_17, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive markers selected from SNP_02 to SNP_17, preferably including at least the donor nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 and/or an Adenine for SNP_04.
- QTL11 is in one aspect located in the region starting at SNP_02 at nucleotide 101 in SEQ ID NO: 2 and ending at SNP_15 at nucleotide 101 in SEQ ID NO: 15.
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_02, SNP_03, SNP_04, SNP_05, SNP_06, SNP_07 and/or SNP_08, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04..
- the (introgression fragment comprising) QTL11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SNP markers selected from SNP_02 to SNP_15, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive markers selected from SNP_02 to SNP_15, preferably including at least the donor nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 and/or an Adenine for SNP_04.
- QTL 11 is in one aspect located in the region starting at SNP_02 at nucleotide 101 in SEQ ID NO: 2 and ending at SNP_12 at nucleotide 101 in SEQ ID NO: 12.
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_02, SNP_03, SNP_04, SNP_05, SNP_06, SNP_07 and/or SNP_08, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04..
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SNP markers selected from SNP_02 to SNP_12, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive markers selected from SNP_02 to SNP_12, preferably including at least the donor nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 and/or an Adenine for SNP_04.
- QTL 11 is in one aspect located in the region starting at SNP_02 at nucleotide 101 in SEQ ID NO: 2 and ending at SNP_10 at nucleotide 101 in SEQ ID NO: 10.
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_03, SNP_04, SNP_05, SNP_06, SNP_07 and/or SNP_08, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the (introgression fragment comprising) QTL11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for 1, 2, 3, 4, 5, 6, 7 or 8 SNP markers selected from SNP_02 to SNP_10, preferably at least 1, 2, 3, 4, 5, 6, 7 or 8 consecutive markers selected from SNP_02 to SNP_10, preferably including at least the donor nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 and/or an Adenine for SNP_04.
- QTL 11 is in one aspect located in the region starting at SNP 01 at nucleotide 101 in SEQ ID NO: 1 and ending at SNP_09 at nucleotide 101 in SEQ ID NO: 09.
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_02, SNP_03, SNP_04, SNP_05, SNP_06, SNP_07 and/or SNP_08, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the (introgression fragment comprising) QTL11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for 1, 2, 3, 4, 5, 6, 7 or 8 SNP markers selected from SNP_01 to SNP_09 or from SNP_02 to SNP_08, preferably at least 1, 2, 3, 4, 5, 6, 7 or 8 consecutive markers selected from SNP_01 to SNP_09 or from SNP_02 to SNP_08, preferably including at least the donor nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 and/or an Adenine for SNP_04.
- QTL11 is in one aspect located in the region starting at SNP_02 at nucleotide 101 in SEQ ID NO: 2 and ending at SNP_06 at nucleotide 101 in SEQ ID NO: 06.
- the (introgression fragment comprising) QTL 11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the (introgression fragment comprising) QTL11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for 1, 2, 3, 4, or 5 SNP markers selected from SNP_02 to SNP_06, preferably at least 1, 2, 3, 4 or 5 consecutive markers selected from SNP_02 to SNP_06, preferably including at least the donor nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP 03 at nucleotide 101 of SEQ ID NO: 3 and optionally a Thymine for SNP_02 and/or an Adenine for SNP_04.
- QTL11 is in another aspect located in the region starting at SNP_02 at nucleotide 101 in SEQ ID NO: 2 and ending at SNP_04 at nucleotide 101 in SEQ ID NO: 04.
- the (introgression fragment comprising) QTL11 comprises at least the donor SNP nucleotide (or donor SNP haplotype) for SNP_02, SNP_03 and/or SNP_04.
- the introgression fragment comprises at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3.
- Fine mapping was carried out as known in the art, in order to determine which subregion of the introgression fragment, i.e. which subregion of the region starting at SNP_01 at nucleotide 101 of SEQ ID NO: 1 and ending at SNP_18 at nucleotide 101 of SEQ ID NO: 18, comprises QTL 11.
- the introgression fragment comprising QTL 11 or a variant of QTL 11 also subregions comprising QTL 11 or a variant of QTL 11 are encompassed herein.
- the subregion of the introgression fragment comprising QTL 11 is in one aspect the region starting at SNP_01 at nucleotide 101 of SEQ ID NO: 1 and ending at SNP_09 at nucleotide 101 of SEQ ID NO: 9, or starting at SNP_02 at nucleotide 101 of SEQ ID NO: 2 and ending at SNP_08 at nucleotide 101 of SEQ ID NO: 8 or ending at SNP_07 at nucleotide 101 of SEQ ID NO: 7, especially the subregion starting at SNP_02 at nucleotide 101 of SEQ ID NO: 2 and ending at SNP_06 at nucleotide 101 of SEQ ID NO: 6 or starting at SNP_02 and ending at SNP_05.
- the subregion comprising QTL11 starts at SNP_02 and ends at SNP_04.
- Any of the above subregions comprise the QTL and preferably comprise at least a Cytosine for SNP_03 at nucleotide 101 of SEQ ID NO: 3 and optionally the donor SNP for SNP_02 and/or SNP_04.
- the region comprising QTL 11 comprises the donor SNP haplotype for SNP_03 and optionally for SNP_02 and/or SNP_04.
- it may also contain the donor SNP for one or more of the other markers, e.g. SNP_01 or SNP_05 to SNP_18.
- QTL11 encompasses in one aspect also functional variants of QTL11 from other wild donors, which comprise the same (donor) SNP haplotype for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or all 18 of the SNP markers SNP_01 to SNP_18 (e.g. for at least SNP_03, or other subgroups of markers as described for QTL11, e.g.
- QTL11 i.e. the introgression fragment comprising the QTL
- QTL11 is present in heterozygous form in a cultivated pepper plant, cell or tissue, especially in sweet pepper.
- QTL11 i.e. the introgression fragment comprising the QTL
- the cultivated pepper plant is an F 1 hybrid, especially an FI hybrid generated by crossing two inbred parent lines, whereby at least one of the parent lines comprises the QTL11 (i.e. the introgression fragment comprising the QTL) preferably in homozygous form.
- the cultivated pepper plant does not comprise any other introgression fragments on chromosome 11 of the pepper genome which affect Aphis gossy ri/ resistance.
- QTL11 (or a variant of QTL11) in heterozygous form also leads to an increase in resistance (reduced susceptibility) against Aphis gossypii, as the QTL is incomplete dominant. It is however preferred that the cultivated pepper plant comprises QTL11 in homozygous form, as hereby the highest resistance level is obtained.
- QTL11 has a positive effect on Aphis gav.sv/w rcsistancc by reducing reproduction, seen e.g. as a reduction in the average number of new nymphs (in e.g. clip-on cage experiments) or as a reduction in population growth (in e.g. population growth experiments) and/or aphid survival, seen e.g. as a reduction in the average number of aphids on plants comprising the QTL compared to the control plants lacking the QTL.
- the effect on resistance of QTL11 or a variant of QTL11 is preferably compared between a line comprising QTL 11 (or a variant) in homozygous form and the recurrent parent or genetic control lacking QTL 11 (or a variant).
- Assays can be used to compare the resistance levels, such as for example free-choice assays or non-choice assays, whereby the reproduction (e.g.
- FIG. 1 shows results of a non-choice assay (see Examples), whereby the reproduction, i.e. the average number of new nymphs was measured in clip-on cages.
- the wild C. baccatum QTL 11 donor plant has the highest level of resistance, as on this plant significantly fewer average (new) nymphs were counted compared to the elite C. annuum line lacking QTL11. However, also the backcross C.
- annuum line (indicated as QTL 11 CH11 Hom(R)), into which QTL 11 was introgressed (and in which QTL 11 is in homozygous form), shows significantly fewer new nymphs than the Elite C. annuum line lacking QTL 11, showing that QTL11 significantly reduces the average number of new nymphs, i.e. reduces aphid reproduction.
- a cultivated Capsicum plant of a domesticated Capsicum species especially a cultivated C. annuum plant
- chromosome 11 comprises an introgression fragment from a wild Capsicum, especially from a wild C. baccatum, whereby the introgression fragment comprises QTL 11 (or a variant), which increases resistance of the plant against Aphis gossypii.
- the other chromosomes are all cultivated genome. That is to say that in one aspect of the invention the cultivated Capsicum plant (especially C. annuum) comprises only one introgression fragment from a wild C.
- the cultivated Capsicum plant which comprises QTL 11 (or a variant) from a wild Capsicum donor, especially a wild C. baccatum donor, may also comprise other introgression fragments, either on chromosome 11 and/or on other chromosomes, e.g. from other wild Capsicum accessions.
- such introgression fragments may also comprise genes or QTLs which increase resistance against Aphis gossypii.
- the plant may comprise an introgression fragment on chromosome 2, conferring resistance against Myzus persicae, as described by Sun et al. (supra).
- the source of the QTL11 i.e. the QTL11 donor
- the source of the QTL 11 is another wild Capsicum accession, especially another Capsicum baccatum accession, comprising the donor haplotype of the SNP markers linked to QTL11, e.g.
- the haplotype of the donor for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, of the SNP markers SNP_01 to SNP_18 linked to QTL11, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, of the SNP markers SNP_02 to SNP_17 linked to QTL11, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, of the SNP markers SNP_02 to SNP_15 linked to QTL11, or for at least 1, 2, 3, 4, 5, 6, 7, 8, or more, of the SNP markers SNP_02 to SNP_12 linked to QTL11, or for at least 1, 2, 3, 4, 5, 6, 7 or 8 of the SNP markers SNP_02 to SNP_10 linked to QTL11, or especially for at least 1, 2, 3, 4, 5, 6, 7 or more of SNP_01 or SNP_02 to SNP_09, or SNP_02 to SNP_08, especially of SNP_02 to SNP_07, or SNP_01 or SNP_02
- accession comprises the SNP donor haplotype for at least 1, 2, 3, 4 or 5 of the SNP markers of SNP_02 to SNP_6 or SNP_02 to SNP_05 or of SNP_02 to SNP_04 linked to QTL11.
- the donor haplotype preferably at least includes a Cytosine for SNP_03 at nucleotide 101 of SEQ ID N: 3, as SNP_03 is the most closely liked marker.
- the QTL 11 from other wild Capsicum accessions is herein also referred to as ‘variant QTL 1 G or ‘functional variant of QTL1 G, as the SNP haplotype linked to QTL11 may not be 100% identical to the SNP haplotype of the wild donor as present in NCIMB 43605 for all of SNP_01 to SNP_18, but only for subgroups thereof, as described above.
- the donor comprises a SNP haplotype which is identical to the haplotype found in NCIMB 43605 for at least 2, 3, 4, 5, 6, 7 or more markers selected from any of the subgroups: for SNP_01 or SNP_02 to SNP_09, or for SNP_01 or SNP_02 to SNP_08, or for SNP_01 or SNP_02 to SNP_07, or for SNP_01 or SNP_01 or SNP_02 to SNP_06, or for SNP_01 or SNP_02 to SNP_05, or for SNP_01 or SNP_02 to SNP_04 or SNP_01 or SNP_02 to SNP_03 or SNP_03 to SNP_04.
- variant QTL 11 can, thus, be identified by the SNP haplotype and by the phenotype it confers, i.e. it also confers enhanced resistance to Aphis gossypii, which can be verified by introgressing the variant QTL 11 (e.g. by marker assisted selection using one or more of the SNP markers) into a susceptible cultivated Capsicum line or variety.
- the cultivated Capsicum plant of the invention may, in addition to QTL11 (or the variant QTL11), comprise one or more other introgression fragments from wild Capsicum accessions in its genome.
- the SNP markers and/or the sequences comprising the SNP markers can be used to identify other wild donors comprising QTL 11 (or a variant thereof), e.g. by identifying a wild Capsicum donor comprising the donor haplotype of the SNP markers linked to the QTL, e.g. the haplotype of the donor for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 (or more, e.g.
- SNP_01 to SNP_18 or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 (or more, e.g. all) of the SNP markers SNP_02 to SNP_17, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 (or more, e.g.
- SNP markers SNP_02 to SNP_15 are all) of the SNP markers SNP_02 to SNP_15, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 of the SNP markers SNP_02 to SNP_12, or for at least 1, 2, 3, 4, 5, 6, 7, 8 of the SNP markers SNP_02 to SNP_10, or especially or for at least 1, 2, 3, 4, 5, 6, 7, 8 of the SNP markers SNP_01 or SNP_02 to SNP_09, or of the SNP markers SNP_01 or SNP_02 to SNP_08, or of the SNP markers SNP_01 or SNP_02 to SNP_07, or of the SNP markers of SNP_01 or SNP_02 to SNP_06, or of the SNP markers SNP_01 or SNP_02 to SNP_05, or of the SNP markers of SNP_01 or SNP_02 to SNP_04, or for SNP_01 or SNP_02 to SNP_03 or for SNP_03 to SNP_
- the above groups of markers in one aspect comprise at least a Cytosine for SNP_03, i.e. the donor SNP for SNP_03 and optionally the donor SNP nucleotide for SNP_02 and/or SNP_04.
- the left and right flanking sequences of the SNP markers provided herein are the genomic DNA sequences of the reference genome of C. annuum, meaning that there may be slight sequence variation in the actual wild donor sequences flanking the SNP markers. Therefore, when herein reference is made to a SNP nucleotide in the SEQ ID NO:s provided herein, e.g. SNP 01 at nucleotide 101 of SEQ ID NO: 1, it is understood that the sequence before and after this SNP nucleotide (i.e. the left and right flanking sequence) may not be 100% identical to the sequences provided, but may be 95%, 96%, 97%, 98% or 99% identical.
- the wild donor nucleotide for SNP 01 is a Guanine at nucleotide 101 of SEQ ID NO: 1 or it is a Guanine at nucleotide 101 of a sequence comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.
- Such a sequence, where 1, 2 or 3 nucleotides may differ in the left or right flanking sequence of the SNP may also be referred to as a ‘variant’ of the SEQ ID NO, i.e. a ‘variant sequence’.
- Sequence identity is preferably determined by pairwise alignment of two sequences using the program Needle of Emboss (default parameters).
- flanking sequence of any SNP marker provided herein can be determined by methods known in the art, such as sequencing, e.g. by amplifying the DNA sequence to the right and left of each SNP, e.g. by PCR, and sequencing the amplification product.
- the deposited seeds are not seeds of a pepper variety, but the seeds can be used to develop many different distinct, uniform and stable pepper varieties (as defined by UPOV) comprising QTL11, preferably in homozygous form.
- Capsicum The genus Capsicum consists of 20 to 27 species, five of which are domesticated: C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens. Phylogenetic relationships between species have been investigated using bio-geographical, morphological, chemosystematic, hybridization, and genetic data. Fruits of Capsicum can vary tremendously in color, shape and size, both between and within species. Chemosystematic studies helped distinguish the difference between varieties and species. Capsicum annuum L. plants are herbaceous plants of the family Solanaceae. The plant reaches about 0.5-1.5 meters (20 to 60 inches).
- Cultivated pepper plants of the species Capsicum annuum include different types of peppers, such as bell peppers, cayenne peppers, paprika, and jalapenos.
- the hot pepper genome (Mexican landrace of Capsicum annuum cv. CM334) has been sequenced by an international group of scientists from Korea, Israel and USA. The sequence was published in Nature Genetics in January 2014 (Kim, S., Park, M., Yeom, S. et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet 46, 270-278, 2014). The official page for this genome sequences is http://peppergenome.snu.ac.kr/. The genome size is estimated to be 3.48 Gb.
- Aphis gossypii is very damaging to many economically important crops, including cotton, aubergine / eggplant, citrus, coffee, melon, okra, peppers, potato, squash and sesame.
- A. gossypii has a worldwide distribution, both in the field and in greenhouses. Economic damage due to A. gossypii is by direct feeding, the excretion of honey dew and virus transmission. The initial symptom of A. gossypii attack is a yellowing of the leaves. As aphids become more numerous, leaves become puckered and curled.
- the inventors have identified a wild Capsicum baccatum accession, which comprises resistance against A. gossypii. Although crossing this accession with Capsicum annuum was extremely difficult, they succeeded with the help of embryo rescue to develop a backcross population. Only 1 out of 22 interspecific crosses was successful with the aid of embryo rescue. Sufficient plants were available for QTL mapping of this BC1F1 population and they found a major QTL on chromosome 11 from the C. baccatum donor accession.
- Figure 1 shows the results of a clip-on cage experiment described in the Examples, showing average numbers of new nymphs.
- the ‘donor’ is the wild C. baccatum donor accession.
- ‘Elite’ is the C. annuum recurrent parent used in the backcrossing, and ‘QTL CHR11 Horn (R)’ is a backcross line into which QTL11 has been crossed into from the donor, and which is homozygous for QTL11.
- the introgression of QTL 11 from the donor into the Elite parent therefore clearly reduces the average number of nymphs, and thus increases resistance against A. gossypii.
- Figure 2 shows the genetic linkage map of chromosome 11 and the position of the markers in centiMorgan (cM).
- ID 1 to ID 18 refer to the SNP markers SNP_01 to SNP_18, the other markers (designated mPP) are in- house markers.
- the interval to which QTL11 has been mapped is indicated as the bar next to the chromosome.
- SNP_01 to SNP_18 are in the QTL 11 interval (longer bar), and SNP_01 to SNP_09 are in the fine-mapped QTL11 interval (shorter bar). This region is homozygous for the wild donor SNP genotype in the cultivated C. annuum seeds deposited herein under accession number NCIMB43605.
- indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
- the indefinite article “a” or “an” thus usually means “at least one”.
- plant includes the whole plant or any parts or derivatives thereof, such as plant organs (e.g., harvested or non-harvested storage organs, tubers, fruits, leaves, seeds, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, ovaries, fruits (e.g., harvested tissues or organs, such as harvested fruits or parts thereof), flowers, leaves, seeds, tubers, bulbs, clonally propagated plants, roots, root-stocks, stems, root tips and the like.
- plant organs e.g., harvested or non-harvested storage organs, tubers, fruits, leaves, seeds, etc.
- plant cells e.g., plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts
- seedlings any developmental stage is included, such as seedlings, immature and mature, etc.
- seed of a plant these either refer to seeds from which the plant can be grown or to seeds produced on the plant, after self-fertilization or cross-fertilization.
- Plant variety is a group of plants within the same botanical taxon of the lowest grade known, which (irrespective of whether the conditions for the recognition of plant breeder’s rights are fulfilled or not) can be defined on the basis of the expression of characteristics that result from a certain genotype or a combination of genotypes, can be distinguished from any other group of plants by the expression of at least one of those characteristics, and can be regarded as an entity, because it can be multiplied without any change.
- the term “plant variety” cannot be used to denote a group of plants, even if they are of the same kind, if they are all characterized by the presence of one or two loci or genes (or phenotypic characteristics due to these specific loci or genes), but which can otherwise differ from one another enormously as regards the other loci or genes.
- a plant defined only by the presence of QTL11 is not a plant variety, as thousands of other genes which define a plant variety are undefined and a plant defined only by the presence of QTL 11 is not uniform and stable for these thousands of genes and the characteristics conferred by these genes.
- QTL11 can be used to develop many different plant varieties, e.g. a sweet pepper variety which is uniform and stable for all its physiological and morphological characteristics such as leaf size or shape, fruit size and color, plant height, etc. and which also comprises QTL11.
- FI, F2, F3, etc. refers to the consecutive related generations following a cross between two parent plants or parent lines. The plants grown from the seeds produced by crossing two plants or lines is called the FI generation. Selfing the FI plants results in the F2 generation, etc.
- F 1 hybrid plant (or F 1 hybrid seed) is the generation obtained from crossing two inbred parent lines.
- FI hybrid seeds are seeds from which FI hybrid plants grow.
- FI hybrids are more vigorous and higher yielding, due to heterosis.
- Inbred lines are essentially homozygous at most loci in the genome.
- a “plant line” or “breeding line” refers to a plant and its progeny.
- the term "inbred line” refers to a plant line which has been repeatedly selfed and is nearly homozygous.
- an “inbred line” or “parent line” refers to a plant which has undergone several generations (e.g. at least 4, 5, 6, 7 or more) of inbreeding, resulting in a plant line with a high uniformity.
- allele(s) means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus.
- alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome.
- loci plural locus on a chromosome.
- One allele is present on each chromosome of the pair of homologous chromosomes.
- a diploid plant species may comprise a large number of different alleles at a particular locus. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous).
- a . gossypii resistance allele or a “ A . gossypii tolerance allele” of the locus QTL11.
- gene means a (genomic) DNA sequence comprising a region (transcribed region), which is transcribed into a messenger RNA molecule (mRNA) in a cell, and an operably linked regulatory region (e.g. a promoter).
- mRNA messenger RNA molecule
- an operably linked regulatory region e.g. a promoter
- Different alleles of a gene are thus different alternatives form of the gene, which may be in the form of e.g. differences in one or more nucleotides of the genomic DNA sequence (e.g. in the promoter sequence, the exon sequences, intron sequences, etc.), mRNA and/or amino acid sequence of the encoded protein.
- locus means a specific place or places or a site on a chromosome where for example a QTL, a gene or genetic marker is found.
- the A. gossypii resistance locus is, thus, the location in the genome of wild or cultivated Capsicum (especially Capsicum annuum), where QTL11 is found.
- the QTL is found on chromosome 11 (using the chromosome assignment of Kim et ah, 2014, supra, and as found at http://peppergenome.snu.ac.kr), i.e. the QTL11 is introgressed into the cultivated genome (i.e. onto chromosome 11) from a wild Capsicum ssp. (also referred to as donor herein).
- QTL quantitative trait locus
- QTL 11 The A. gossypii resistance resistance conferring quantitative trait locus is named QTL 11 herein.
- “Cultivated Capsicum genome” or “cultivated pepper genome” or “physical position on the cultivated pepper genome” and “chromosome 11” refers in one aspect to the physical genome of cultivated pepper. Genome sequences of variety CM334 are found on the world wide web at http://peppergenome.snu.ac.kr and the physical chromosomes and the physical position on the chromosomes can be determined for each SNP marker. The physical position will be different in different versions of the genome, e.g. in the genome of CM334vl.55 or in CM334vl.6.
- “Physical distance” between loci (e.g. between molecular markers and/or between phenotypic markers) on the same chromosome is the physical distance expressed in bases or base pairs (bp), kilo bases or kilo base pairs (kb) or megabases or mega base pairs (Mb).
- “Genetic distance” between loci (e.g. between molecular markers and/or between phenotypic markers) on the same chromosome is measured by frequency of crossing-over, or recombination frequency (RF) and is indicated in centimorgans (cM).
- RF recombination frequency
- cM centimorgans
- One cM corresponds to a recombination frequency of 1%. If no recombinants can be found, the RF is zero and the loci are either extremely close together physically or they are identical. The further apart two loci are, the higher the RF.
- “Introgression fragment” or “introgression segment” or “introgression region” refers to a chromosome fragment (or chromosome part or region) which has been introduced into another plant of the same or related species by crossing or traditional breeding techniques, such as backcrossing, i.e. the introgressed fragment is the result of breeding methods referred to by the verb “to introgress” (such as backcrossing).
- wild Capsicum accessions can be used to introgress fragments of the wild genome (donor) into the genome of cultivated Capsicum plants of a domesticated species, especially C. annuum.
- Such a cultivated Capsicum plant thus has a “genome of cultivated Capsicum, especially C.
- annuum but comprises in the genome a fragment of a wild Capsicum donor, e.g. an introgression fragment of a wild Capsicum genome, such as Capsicum baccatum.
- a cultivated Capsicum plant especially C. annuum, C. baccatum, C. frutescens, C. chinense or C. pubescens
- a genome of cultivated Capsicum is provided herein comprising a genome of cultivated Capsicum, and in that genome one introgression fragment on chromosome 11 from a wild Capsicum donor accession which confers A.
- introgression fragment never includes a whole chromosome, but only a part of a chromosome, and that the introgression fragment is characterized by the donor SNP markers or donor SNP haplotype of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SNP markers, especially consecutive SNP markers.
- the introgression fragment can be large, e.g.
- a chromosome even three quarter or half of a chromosome, but is preferably smaller, such as less than 200 Mb, such as about 197 Mb or less, about 150Mb or less, about 125 Mb or less, about 100 Mb or less, about 80 Mb or less, about 50 Mb or less, about 25 Mb or less, about 20Mb or less, about 10 Mb or less, about 8 Mb or less, about 7 Mb or less, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2.5 Mb or 2 Mb or less, about 1 Mb (equals 1.000.000 base pairs) or less, or about 0.5 Mb (equals 500.000 base pairs) or less, such as about 350.000 bp, 200.000 bp (equals 200 kilo base pairs) or less, about 100.000 bp (100 kb) or less, about 50.000 bp (50 kb) or less, about 25.000 bp
- pepper plant or “pepper” is a plant of genus Capsicum or parts thereof (e.g. fruits).
- Pepper includes all kinds of peppers, such as hot/pungent peppers and non-pungent peppers (sweet pepper). The term encompasses wild accessions and domesticated peppers.
- Domesticated pepper refers to the species Capsicum annuum L., Capsicum chinense Jacq., Capsicum frutescens L., Capsicum baccatum L., and Capsicum pubescens Ruiz & Pav.
- cultivar or “cultivated” plant or “cultivated Capsicum plant” is used herein to denote a plant having a biological status other than a "wild" status, which "wild" status indicates the original non-cultivated, non- domesticated, or natural state of a plant or accession, and the term cultivated does not include such wild, or weedy plants.
- cultivar does include material with good agronomic characteristics, such as breeding material, research material, breeding lines, elite breeding lines, synthetic population, hybrid, founder stock/base population, inbred lines, cultivars (open pollinated of hybrid cultivar), segregating population, mutant/genetic stock, and advanced/improved cultivar.
- the term cultivar also includes landraces, i.e.
- cultivars include cultivated varieties that belong to the species Capsicum annuum, Capsicum chinense, Capsicum frutescens, Capsicum baccatum and Capsicum pubescens.
- Capsaicin is the active component of chili peppers, which are plants belonging to the genus Capsicum. It is an irritant for mammals, including humans, and produces a sensation of burning. The amount of capsaicin significantly varies between different cultivated Capsicum species, including Capsicum annuum.
- the “Scoville scale” is a measurement of the spicy heat (or piquancy / pungency) of pepper.
- the number of Scoville heat units (SHU) indicates the amount of capsaicin present: An alcohol extract of the capsaicin oil from a measured amount of dried pepper is added incrementally to a solution of sugar in water until the "heat” is just detectable by a panel of (usually five) tasters; the degree of dilution gives its measure on the Scoville scale.
- spice heat can be measured by using high performance liquid chromatography (HPLC). This method yields results in American Spice Trade Association (ASTA) pungency units. HPLC methods are well known in the art. See, e.g., a description of DIONEX.
- the isocratic analytical system consisted of a pump, autosampler, thermostatic chamber, a four channel CoulArray® detector and an UV/vis detector placed before the array.
- LC Conditions Column: MD-150 (3 x 150mm; 3pm), Mobile Phase: 50mM Ammonium Acetate, pH 4.4, with acetic acid; 45% Acetonitrile, Flow Rate: 0.8 mL/min, Temperature: Ambient, Injection Volume: 20 m ⁇ , Detectors and Conditions: Electrochemical Detector: Model 5600A, CoulArray, with Model 5010 Analytical Cell, Applied Potentials: +150, +450mV vs. Pd, UV Detector: Standard UV detector, Wavelength: 235 and 280nm. A measurement of one part capsaicin per million corresponds to about 15 Scoville units. However, the Scoville method is preferred when determining the piquancy / pungency of pepper.
- “Uniformity” or “uniform” relates to the genetic and phenotypic characteristics of a plant line or variety. Inbred lines are genetically highly uniform as they are produced by several generations of inbreeding. Likewise, and the F 1 hybrids which are produced from such inbred lines are highly uniform in their genotypic and phenotypic characteristics and performance.
- Aphis gossypii resistance allele refers to an allele found at the locus QTL11 introgressed into cultivated Capsicum of a domesticate Capsicum species (onto cultivated Capsicum chromosome 11) from a wild donor.
- the term thus, also encompasses alleles obtainable from other wild Capsicum donor accessions, which e.g. comprise the same donor haplotype for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more (or all, or all but 1, 2, or 3) of the SNP markers linked to the QTL11, e.g. to the large QTL11 region or to the fine- mapped region or subregions thereof.
- the plant, plant line or variety comprises a significantly reduced susceptibility (increased resistance) to Aphis gossypii than the control lacking the QTLs, preferably the genetic control.
- the allele found at the same locus on chromosome 11 is herein referred to as “wild type” allele (wt).
- wild type allele wt
- the genotype or haplotype of the SNP markers provided herein, which are physically linked to the allele, is also indicative of the wild type allele or of the QTL being in homozygous or heterozygous form.
- the genotype of SNP_01 indicative of QTL11 is ‘GT’ ( QTLll/wt ) or ‘GG’ ( QTL11/QTL11 ), while the genotype indicative ofthe wild type is ‘TT’ (wt/wt), the genotype of SNP_02 indicative of QTL11 is ‘TC’ ( QTLll/wt ) or ‘TT’ ( QTL11/ QTL11 ), while the genotype indicative ofthe wild type is ‘CC’ ⁇ wt/wt), etc.
- the haplotype for SNP_01 and SNP_02 indicative of QTL11 being in homozygous form is GG-TT.
- SNP marker refer herein to single nucleotide polymorphisms of a genomic sequence linked to QTL11, whereby a specific nucleotide (e.g. for SNP 01 a Guanine at nucleotide 101 of SEQ ID NO: 1, or a Guanine at nucleotide 101 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 1), or a sequence comprising the specific nucleotide, is linked to the QTL.
- a specific nucleotide e.g. for SNP 01 a Guanine at nucleotide 101 of SEQ ID NO: 1, or a Guanine at nucleotide 101 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 1
- SNP genotype or ‘ SNP nucleotide ’ of the plant or plant part
- SNP_01 may be ‘G’ (haploid, on one chromosome) or ‘GG’ (diploid, on both chromosomes).
- Markers SNP_01 to SNP_18, or subgroups e.g.
- a DNA sequence comprising a SNP includes the plus strand and/or the minus (complementary) strand of a double stranded DNA, e.g. a marker assay may be designed to detect a SNP in the plus or in the minus strand of the genomic DNA.
- the ‘haplotype ’or “haploid genotype” refers to the haploid genotype of several genetic loci in a plant, especially of several SNP markers or several sequences comprising the SNP markers.
- the SNP haplotype may thus be the haploid genotype of at least 2, 3, 4, 5, 6 or more (e.g. all 18) SNP markers of SNP_01 to SNP_18 (or of the sequences comprising the SNP markers) or of any subgroups thereof.
- the plant comprising QTL11 may comprise a ‘G’ for SNP 01 at nucleotide 101 in SEQ ID NO: 1 (or at nucleotide 101 of a sequence which is at least 95% identical to SEQ ID NO: 1), a ‘T’ for SNP_02 at nucleotide 101 in SEQ ID NO: 2 (or at nucleotide 101 of a sequence which is at least 95% identical to SEQ ID NO: 2), a ‘C’ for SNP_03 at nucleotide 101 of SEQ ID NO: 3, it thus has the SNP haplotype G-T-C for SNP_01 to SNP_03, which is the SNP haplotype of SNP_01 to SNP_03 of the wild donor (also referred to as donor SNP haplotype).
- a genetic element, an introgression fragment, or a QTL or a gene or allele conferring a trait is said to be “obtainable from” or can be “obtained from” or “derivable from” or can be “derived from” or “as present in” or “as found in” a plant or seed or tissue or cell if it can be transferred from the plant or seed in which it is present into another plant or seed in which it is not present (such as a line or variety) using traditional breeding techniques without resulting in a phenotypic change of the recipient plant apart from the addition of the trait conferred by the genetic element, locus, introgression fragment, gene or allele.
- the terms are used interchangeably and the genetic element, locus, introgression fragment, gene or allele can thus be transferred into any other genetic background lacking the trait. Not only seeds deposited and comprising the genetic element, locus, introgression fragment, gene or allele can be used, but also progeny/descendants from such seeds which have been selected to retain the genetic element, locus, introgression fragment, gene or allele, can be used and are encompassed herein, such as commercial varieties developed from the deposited seeds or from descendants thereof.
- Whether a plant (or genomic DNA, cell or tissue of a plant) comprises the same genetic element, locus, QTL, introgression fragment, gene or allele as obtainable from the deposited seeds can be determined by the skilled person using one or more techniques known in the art, such as phenotypic assays, whole genome sequencing, molecular marker analysis, trait mapping, chromosome painting, allelism tests and the like, or combinations of techniques.
- a “Variant” or “orthologous” sequence or a “variant QTL11” or a “functional variant” of QTL11 refers to a Aphis gossypii resistance conferring QTL (QTL11), or an introgression fragment comprising the QTL, which is derived from a different wild donor plant than the QTL11 present in NCIMB43605.
- Such a variant QTL can be identified as having the same SNP haplotype as the QTLs present in NCIMB43605 for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more markers (preferably consecutive markers) selected from SNP_01 to SNP_18, especially selected from SNP_01 to SNP_09, or from SNP_02 to SNP_08, or from SNP_01 or SNP_02 to SNP_06, or from SNP_02 to SNP_04, for a variant of QTL11.
- a plant comprising a variant QTL 11 may comprise a SNP haplotype G-T-C-A-A-G-C-G-G for SNP_01 to SNP_09, i.e.
- a ‘G’ for SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence which is at least 95% identical to SEQ ID NO: 1)
- a ‘T’ for SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence which is at least 95% identical to SEQ ID NO: 2)
- a ‘C’ for SNP_03 at nucleotide 101 of SEQ ID NO: 3 or at nucleotide 101 of a sequence which is at least 95% identical to SEQ ID NO: 3
- an ‘A’ for SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence which is at least 95% identical to SEQ ID NO: 4)
- an ‘A’ for SNP_05 at nucleotide 101 in SEQ ID NO: 5 (or at nucleotide 101 of a sequence which is at least 95% identical
- An “increased resistance against Aphis gossypii ” or a “significantly increased Aphis gossypii resistance” or a “reduced susceptibility to Aphis gossypii ” refers to a cultivated Capsicum plant, plant line, hybrid or variety comprising an introgression fragment on chromosome 11, comprising QTL11 (or a variant QTL11), having (due to the QTLs) a (statistically significant) reduced number of new nymphs and/or of adults being present on the plant line in no-choice tests (such as a clip-on cage test) compared to the susceptible control and/or reducing aphid population growth in free-choice tests, compared to the susceptible control plant, such as the recurrent parent lacking QTL11 or a genetic control. See also the Examples.
- “Clip-on cage assay” or “clip-on cage test” refers to an assay as e.g. described in the Examples, whereby several plants of a plant comprising QTL11 and several plants of a susceptible control lacking QTL11 (preferably the genetic control) are grown. Per plant e.g. two clip-on cages are placed onto the leaves of each plant. In each clip-on cage a number of adults, e.g. 4-5 Aphis gossypii adults, are placed and left for 24 hours. Then all insects are removed, except that in each clip-on cage e.g. only 5 one-day old nymphs are left. These nymphs are left inside the cage for a specified period (e.g.
- Aphid population growth assay refers to a free choice assay wherein e.g. 6 to 8 week old pepper plants of plants comprising QTL11 and control plants are grown in a greenhouse (e.g. in a complete randomized design) and all plants are covered together with an insect proof cover. Each plant is infested with a leaf disc harbouring at least 50 insects of Aphis gossypii. The insect populations are left to grow for e.g. 10 days (dpi) and the plants are then assessed visually on a scale of e.g.
- Control plant is a cultivated Capsicum genotype, breeding line, hybrid or variety lacking the introgression fragment.
- the control plant should be of the same type as the plant comprising the introgression fragment(s), e.g. cultivated C. annuum sweet pepper when the introgression fragment was introgressed into cultivated C. annuum sweet pepper.
- the susceptible parent line into which the QTL 11 is introgressed also referred to as the recurrent parent
- the recurrent parent is a suitable control.
- Genetic control is a control plant of cultivated Capsicum genotype, breeding line, variety or hybrid which has the same or very similar cultivated genome as the plant comprising the introgression on chromosome 11 except that it lacks the introgressions on chromosome 11, i.e. chromosome 11 is “wild type” cultivated Capsicum genome. This is for example a backcross line in the backcrossing program which does not contain the introgression fragments.
- marker assay refers to a molecular marker assay which can be used to test whether on cultivated Capsicum chromosome 11 an introgression from a wild donor is present which introgression fragment comprises QTL11 (or whether a wild Capsicum accession comprises QTL11, or a variant thereof, in its genome), by determining the genotype or haplotype of any one or more markers linked to the QTL11, e.g. the genotype or haplotype of one or more SNP markers selected from SNP_01 to SNP_18, or selected from SNP_01 to SNP_09, or from SNP_02 to SNP_08, or from SNP_01 or SNP_02 to SNP_06, or from SNP_02 to SNP_04.
- “Flanking markers” are markers which are on either side of the QTL, i.e. the QTL is located on the chromosomal region in-between the flanking markers, e.g. the QTL11 (or a variant QTL11) is in one aspect located in between SNP 01 at nucleotide 101 of SEQ ID NO: 1 and SNP 18 at nucleotide 101 of SEQ ID NO: 18, or between other flanking markers described herein.
- QTL 11 (or a variant thereof) is located between SNP_01 or SNP_02 and SNP_09, or between SNP_01 or SNP_02 and SNP_08, or between SNP_01 or SNP_02 and SNP_07, or between SNP_01 or SNP_02 and SNP_06, or between SNP_01 or SNP_02 and SNP_05, or between SNP_01 or SNP_02 and SNP_04.
- SNP markers i.e. SNP_01 to SNP_18 for chromosome 11, are located in the given order on the introgression fragment.
- “Consecutive” markers refers to markers in the same consecutive order, so e.g. two consecutive markers may be SNP_01 and SNP_02; SNP_02 and SNP_03; SNP_03 and SNP_04, etc. and three consecutive markers may be SNP_01 and SNP_02 and SNP_03; SNP_02 and SNP_03 and SNP_04; etc.
- Average refers herein to the arithmetic mean and both terms are used interchangeably.
- the term “average” or “mean” thus refers to the arithmetic mean of several measurements.
- arithmetic means of at least 8, 9, 10, 15, 20, 30, 40, 50 or more plants (or plant parts) are measured, preferably in randomized experimental designs with several replicates and suitable control plants grown under the same conditions in the same experiment.
- Statistically significant or “statistically significantly” different or “significantly” different refers to a characteristic of a plant line or variety that, when compared to a suitable control (e.g.
- a “recombinant chromosome” refers to a chromosome having a new genetic makeup arising through crossing- over between homologous chromosomes, e.g. a “recombinant chromosome 11”, i.e. a chromosome 11 which is not present in either of the parent plants and arose through a rare double crossing-over event between homologous chromosomes of a chromosome 11 pair.
- a recombinant Capsicum annuum chromosome 11 comprising an introgression from a wild C. baccatum donor accession, which comprises a QTL that enhances A. gossypii resistance.
- Targeted gene/genome editing refers to methods whereby a target gene, such as the causal gene (or candidate gene) underlying QTL 11, is modified by e.g. CRISPR based methods (Crispr-Cas9 or Crispr-Cpfl, etc.), TALENs or other methods known in the art.
- Backcrossing refers to a breeding method by which a (single) trait, such as a A. gossypii resistance QTL, can be transferred from a (generally inferior) genetic background (e.g. a wild Capsicum accession, also referred to as “donor”) into a different (generally superior) genetic background (also referred to as “recurrent parent”), e.g. cultivated Capsicum of a domesticated Capsicum species.
- a cross e.g.
- an FI plant obtained by crossing a wild Capsicum with a cultivated Capsicum; or an F2 plant or F3 plant, etc., obtained from selfing the FI) is “backcrossed” to the parent with the different (generally superior) genetic background, e.g. to the cultivated parent. After repeated backcrossing, the trait of the first (generally inferior) genetic background will have been incorporated into the different (generally superior) genetic background.
- Marker assisted selection is a process of using the presence of molecular markers, which are genetically and physically linked to a particular locus or to a particular chromosome region (e.g. introgression fragment or subfragment), to select plants for the presence of the specific locus or region (introgression fragment or subfragment).
- molecular markers genetically and physically linked to a A. gossypii resistance QTL, can be used to detect and/or select plants comprising the QTL on chromosome 11. The closer the genetic linkage of the molecular marker to the locus (e.g.
- a marker “within 15 cM, 10 cM or 7 cM or within 5 cM, 3 cM, 2 cM, or 1 cM” of another marker refers to a marker which genetically maps to within the 15cM, 10 cM, 7cM or 5cM, 3 cM, 2 cM, or 1 cM region flanking the marker (i.e. either side of the marker).
- a marker within 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4Mb, 0.3Mb, 0.2Mb, 0.1 Mb, 50kb, 20kb, lOkb, 5kb, 2kb, lkb or less of another marker refers to a marker which is physically located within the 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4Mb, 0.3Mb, 0.2Mb, 0.1 Mb, 50kb, 20kb, lOkb, 5kb, 2kb, lkb or less, of the genomic DNA region flanking the marker (i.e. either side of the marker).
- LOD-score (logarithm (base 10) of odds) refers to a statistical test often used for linkage analysis in animal and plant populations. The LOD score compares the likelihood of obtaining the test data if the two loci (molecular marker loci and/or a phenotypic trait locus) are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD scores favor the presence of linkage and a LOD score greater than 3.0 is considered evidence for linkage. A LOD score of +3 indicates 1000 to 1 odds that the linkage being observed did not occur by chance.
- “Vegetative propagation”, “vegetative reproduction” or “clonal propagation” are used interchangeably herein and mean the method of taking part of a plant and allowing that plant part to form at least roots where plant part is, e.g., defined as or derived from (e.g. by cutting of) leaf, pollen, embryo, cotyledon, hypocotyl, cells, protoplasts, meristematic cell, root, root tip, pistil, anther, flower, shoot tip, shoot, stem, fruit, petiole, etc.
- vegetative propagation When a whole plant is regenerated by vegetative propagation, it is also referred to as a vegetative propagation.
- Cell culture or “tissue culture” refers to the in vitro culture of cells or tissues of a plant.
- Regeneration refers to the development of a plant from cell culture or tissue culture or vegetative propagation.
- Non-propagating cell refers to a cell which cannot be regenerated into a whole plant.
- Transgene or “chimeric gene” refers to a genetic locus comprising a DNA sequence, such as a recombinant gene, which has been introduced into the genome of a plant by transformation, such as Agrobacterium mediated transformation.
- a plant comprising a transgene stably integrated into its genome is referred to as “transgenic plant”.
- isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.
- sequence When referring to a “sequence” herein, it is understood that the molecule having such a sequence is referred to, e.g. the nucleic acid molecule.
- a "host cell” or a "recombinant host cell” or “transformed cell” are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, having been introduced into said cell.
- the host cell is preferably a plant cell or a bacterial cell.
- the host cell may contain the nucleic acid as an extra-chromosomally (episomal) replicating molecule, or comprises the nucleic acid integrated in the nuclear or plastid genome of the host cell, or as introduced chromosome, e.g. minichromosome.
- Sequence identity and “sequence similarity” can be determined by alignment of two peptide (protein, amino acid) or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as "substantially identical” or “substantial identity” when they are optimally aligned by for example the programs GAP or BESTFIT or the Emboss program “Needle” (using default parameters, see below) share at least a certain minimal percentage of sequence identity (as defined further below). These programs use the Needleman and Wunsch global alignment algorithm for aligning two sequences, over their entire length, maximizing the number of matches and minimizing the number of gaps.
- sequence similarity or identity may be determined by searching against databases (e.g.
- EMBL, GenBank by using commonly known algorithms and output formats such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity.
- SNP nucleotide or SNP genotype at a specific nucleotide position e.g. at nucleotide 101 of SEQ ID NO: 1, “or at nucleotide 101 of a sequence comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to the SEQ ID NO” or “or at nucleotide 101 of a sequence which is at least 95% identical to the SEQ ID NO”, this means that the SNP nucleotide or SNP genotype is present in a ‘variant sequence’ at a nucleotide corresponding to (or equivalent to) the same nucleotide (e.g.
- nucleotide 101 of SEQ ID NO: 1 in the variant sequence, i.e. in a sequence comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to the mentioned SEQ ID NO. Pairwise alignment if the two sequences can be used to identify the nucleotide corresponding to (or equivalent to) the indicated SNP.
- Stringent hybridisation conditions can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH.
- the Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe.
- stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency.
- Stringent conditions for RNA-DNA hybridisations are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.
- Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g. lOOnt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions.
- “Fine-mapping” refers to methods by which the position of a QTL can be determined more accurately (narrowed down) and/or by which the size of the introgression fragment comprising the QTL is reduced.
- Near Isogenic Lines for the QTL QTL-NILs
- Such lines can then be used to map on which fragment the QTL is located and to identify a line having a shorter introgression fragment comprising the QTL.
- sub-fragments of the introgression fragment for QTL 11 can be identified which comprises the QTL, but which are shorter than the fragment found in the deposited seeds (NCIMB43605), and which consequently lack one or more of the SNP markers of the introgression fragment, especially on either side of the fragment.
- one or more markers on one side of the fragment may not be from the donor / may not be part of the introgression fragment (e.g. SNP_18, SNP_17, etc. towards the beginning of the chromosome, see Figure 2, may be missing and/or one or e.g. SNP_01, SNP_02, etc. towards the end of the chromosome, see Figure 2, may be missing).
- a sub-fragment comprising or retaining QTL11 may comprise less than the 18 donor SNP markers, such as only 2, 3, 4, 5 or 6 consecutive markers selected from SNP_01 to SNP_9.
- the sub-fragment retaining QTL11 comprises SNP_03 and optionally 1, 2, 3 or more markers either side of SNP_03, which is the peak marker.
- the fragment may comprise 6 consecutive markers such as SNP_01 to SNP_06, or SNP_02 to SNP_07, etc. and comprises the QTL11 or variant thereof (conferring reduced susceptibility to A. gossypii, at least when in homozygous form).
- “Candidate gene” is the gene which is assumed to underly and to be causal of the trait conferred by the QTL 11.
- the candidate gene can be found by fine-mapping and looking at the genes present in the narrowed-down region, for example by sequencing the region.
- the candidate gene underlying QTL 11 can, once known, be modified by e.g. mutagenesis techniques (using e.g. chemical or radiation to induce mutations) or by targeted gene editing techniques, etc. to make e.g. a plant comprising a mutant allele of the target gene (such as an allele having reduced expression or no expression of the gene, or an allele encoding a reduced function or loss of function protein) or to increase expression by modifying one or more regulatory sequences of the gene. Also transgenic plants can be made.
- TILLING Targeting Induced Local Lesions IN Genomes
- a ‘TILLING mutant’ refers to a plant which comprises a mutant allele of the target gene, leading e.g. to a change in gene expression or a change in activity of the encoded protein.
- the present invention relates to a cultivated Capsicum plant of a domesticated Capsicum species, especially C. annuum, comprising a QTL on chromosome 11, introgressed from a wild Capsicum donor, which confer an increased resistance / reduced susceptibility against Aphis gossypii compared to a (control) plant lacking the QTL (e.g. the recurrent parent or genetic control).
- a cultivated Capsicum plant of a domesticated Capsicum species, especially C. annuum comprising a QTL on chromosome 11, introgressed from a wild Capsicum donor, which confer an increased resistance / reduced susceptibility against Aphis gossypii compared to a (control) plant lacking the QTL (e.g. the recurrent parent or genetic control).
- the increased A. gossypii resistance is conferred by an introgression fragment on cultivated Capsicum chromosome 11 (comprising QTL
- the introgression fragment is from a donor accession of Capsicum baccatum var. baccatum having small, pungent fruits, but it can also be from a different donor (e.g. a newly identified donor comprising the same SNP haplotype as the donor identified herein for at least 3, 4, 5, 6, 7, 8, 9, 10 or more of the SNP markers linked to the QTL), as described elsewhere herein. Seeds comprising an introgression fragment from a wild donor comprising QTL 11 and comprising donor markers for SNP 01 to SNP 18 to identify the introgression fragment comprising the QTL11 have been deposited.
- introgression fragment on chromosome 11 comprising an A. gossypii resistance QTL this encompasses various sizes of introgression fragments, e.g. the fragment as found in NCIMB43605, or derived from another wild donor comprising the SNP genotype and/or haplotype of the donor for all SNP markers (SNP_01 to SNP_18), but also smaller introgression fragments (sub-fragments) which comprise the QTL11 (or a variant) but which comprise the SNP haplotype indicative of the QTL with fewer SNP markers, e.g.
- Such smaller fragments are thus smaller introgression fragments, which comprise QTL 11 (or a variant thereof) and which lack the SNP donor genotype of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of markers SNP_01 to SNP_18, where however the if agment retains QTL11 or a variant thereof, i.e. it still confers increased resistance against A. gossypii (compared to the control, e.g. the genetic control) e.g. when the introgression fragment is in homozygous form in the cultivated Capsicum genome and also when the introgression fragment is in heterozygous form, although then the effect is less.
- QTL11 was initially mapped to lie om chromosome 11 in between SNP_01 and SNP_18, as shown in Figure 1 (long bar). Later QTL 11 was fine mapped to he in between SNP_01 and SNP_09, as shown in Figure 1 (short bar), with SNP_03 being the peak marker.
- QTL 11 lies in one aspect in between SNP_01 and SNP_09, or in between SNP_02 and SNP_09, or in between SNP_01 and SNP_08, or in between SNP_02 and SNP_08, or in between SNP_01 and SNP_07, or in between SNP_02 and SNP_07, or in between SNP_01 and SNP_06, in between SNP_02 and SNP_06, or in between SNP_01 and SNP_05, in between SNP_02 and SNP_05, or in between SNP_01 and SNP_04, in between SNP_02 and SNP_04, or in between SNP_02 and SNP_03 or in between SNP_03 and SNP_04.
- the large introgression fragment (e.g. present in the deposited seeds) and comprising the donor SNP haplotype for all of SNP_01 to SNP_18 can, therefore, be reduced in size to retain QTL 11 (or a variant thereof), but lacking the other regions either side of the QTL 11.
- a cultivated Capsicum plant comprising an introgression fragment from a wild Capsicum donor, wherein the introgression fragment comprises QTL11, or a variant thereof, and wherein the introgression fragment comprises QTL11 and all or part of the region starting at SNP_01 of chromosome 11 and ending at SNP 18 of chromosome 11, preferably wherein the introgression fragment comprises QTL11 and all or part of the region starting at SNP_01 or SNP_02 and ending at SNP_09, or wherein the introgression fragment comprises QTL11 and all or part of the region starting at SNP_01 or SNP_02 and ending at SNP_08, or wherein the introgression fragment comprises QTL11 and all or part of the region starting at SNP_01 or SNP_02 and ending at SNP_07, or wherein the introgression fragment comprises QTL 11 and all or part of the region starting at SNP_01 or SNP_02 and ending at SNP_06, or wherein the introgression fragment comprises QTL 11 and all or part of the region starting at S
- all or part of the region starting at SNP_01 (at nucleotide 101 of SEQ ID NO: 1) of chromosome 11 and ending at SNP_18 (at nucleotide 101 of SEQ ID NO: 18) of chromosome 11 is, in one aspect, from a wild donor of Capsicum and comprises QTL 11 or a variant thereof.
- the sub-regions containing QTL 11 are the regions selected from the region starting at SNP_01 or SNP_02 and ending at SNP_09, starting at SNP_01 or SNP_02 and ending at SNP_08, starting at SNP_01 or SNP_02 and ending at SNP_07, starting at SNP_01 or SNP_02 and ending at SNP_06, starting at SNP_01 or SNP_02 and ending at SNP_05, starting at SNP_01 or SNP_02 and ending at SNP_04.
- sub-fragment comprising QTL 11 may in one aspect e.g. comprise the donor SNP haplotype of at least 2, 3, 4 or more (or all) consecutive markers SNP_01 or SNP_02 to SNP_09, or SNP_01 or SNP_02 to SNP_08, or SNP_01 or SNP_02 to SNP_07, or SNP_01 or SNP_02 to SNP_06, or SNP_01 or SNP_02 to SNP_05, or SNP_01 or SNP_02 to SNP_04.
- the sub-fragments comprise at least the donor SNP for SNP_03 (i.e. a Cytosine at nucleotide 101 of SEQ ID NO: 3).
- QTL11 (or a variant thereof) is located in-between marker SNP 01 at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1) and marker SNP_18 at nucleotide 101 of SEQ ID NO: 18 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 18).
- all (or less, e.g. 17, 16, 15, 14, 13, 12, 11, 10 or less, e.g. 9, 8, 7, 6, 5, 4 or 3 SNP markers, especially consecutive SNP markers) of SNP_01 to SNP_18 markers indicative of QTL 11 have the SNP haplotype of the donor introgression, i.e.
- SNP 01 comprises an Guanine at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1)
- SNP_02 comprises a Thymine at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 2)
- SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3)
- SNP_04 comprises a Adenine at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 4)
- SNP_05 comprises a Adenine at nucleotide 101 of SEQ ID NO: 5 (or at nucleotide 101 of a sequence comprising at least
- all (or less, e.g. 8, 7, 6, 5, 4, 3 or 2 SNP markers, especially consecutive SNP markers) of SNP_01 or SNP_02 to SNP_09 markers indicative of QTL11 have the SNP haplotype of the donor introgression, i.e.
- SNP_01 comprises an Guanine at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1)
- SNP_02 comprises a Thymine at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 2)
- SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3)
- SNP_04 comprises a Adenine at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 4)
- SNP_05 comprises a Adenine at nucleotide 101 of SEQ ID NO: 5 (or at nucleotide 101 of a sequence comprising at
- At least SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3).
- all (or less, e.g. 7, 6, 5, 4, 3 or 2 SNP markers, especially consecutive SNP markers) of SNP_01 or SNP_02 to SNP_08 markers indicative of QTL11 have the SNP haplotype of the donor introgression, i.e.
- SNP_01 comprises an Guanine at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1)
- SNP_02 comprises a Thymine at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 2)
- SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3)
- SNP_04 comprises a Adenine at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 4)
- SNP_05 comprises a Adenine at nucleotide 101 of SEQ ID NO: 5 (or at nucleotide 101 of a sequence comprising at
- all (or less, e.g. 7, 6, 5, 4, 3 or 2 SNP markers, especially consecutive SNP markers) of SNP_01 or SNP_02 to SNP_07 markers indicative of QTL11 have the SNP haplotype of the donor introgression, i.e.
- SNP_01 comprises an Guanine at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1)
- SNP_02 comprises a Thymine at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 2)
- SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3)
- SNP_04 comprises a Adenine at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 4)
- SNP_05 comprises a Adenine at nucleotide 101 of SEQ ID NO: 5 (or at nucleotide 101 of a sequence comprising at
- all (or less, e.g. 6, 5, 4, 3 or 2 SNP markers, especially consecutive SNP markers) of SNP_01 or SNP_02 to SNP_06 markers indicative of QTL11 have the SNP haplotype of the donor introgression, i.e.
- SNP_01 comprises an Guanine at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1)
- SNP_02 comprises a Thymine at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 2)
- SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3)
- SNP_04 comprises a Adenine at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 4)
- SNP_05 comprises a Adenine at nucleotide 101 of SEQ ID NO: 5 (or at nucleotide 101 of a sequence comprising at
- all (or less, e.g. 6, 5, 4, 3 or 2 SNP markers, especially consecutive SNP markers) of SNP_01 or SNP_02 to SNP_05 markers indicative of QTL11 have the SNP haplotype of the donor introgression, i.e.
- SNP_01 comprises an Guanine at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1)
- SNP_02 comprises a Thymine at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 2)
- SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3)
- SNP_04 comprises a Adenine at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 4)
- SNP_05 comprises a Adenine at nucleotide 101 of SEQ ID NO: 5 (or at nucleotide 101 of a sequence comprising at
- all (or less, e.g. 3 or 2 or 1 of the SNP markers, especially consecutive SNP markers) of SNP_01 or SNP_02 to SNP_04 markers indicative of QTL11 have the SNP haplotype of the donor introgression, i.e.
- SNP_01 comprises an Guanine at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1)
- SNP_02 comprises a Thymine at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 2)
- SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3)
- SNP_04 comprises a Adenine at nucleotide 101 of SEQ ID NO: 4 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 4).
- At least SNP_03 comprises a Cytosine at nucleotide 101 of SEQ ID NO: 3 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 3).
- QTL 11 (or a variant) is located in between flanking markers SNP_01 and SNP_09, or in between SNP_02 and SNP_09, or in between flanking markers SNP_02 and SNP_08, or in between flanking markers SNP_01 and SNP_08, or in between flanking markers SNP_01 and SNP_07, or in between flanking markers SNP_02 and SNP_07, or in between flanking markers SNP_01 and SNP_06, or in between flanking markers SNP_02 and SNP_06, or in between flanking markers SNP_02 and SNP_06, or in between flanking markers SNP_02 and SNP_05, or in between flanking markers SNP_01 and SNP_05, or in between flanking markers SNP_02 and SNP_
- flanking markers may comprise the donor SNP genotype or the recurrent parent SNP genotype.
- the genotype of the SNP markers in between the flanking markers is in one aspect the donor SNP genotype or haplotype.
- at least SNP_03 comprises the donor nucleotide.
- the introgression fragment may comprise the donor SNP haplotype for all SNP markers, or only for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the SNP markers selected from SNP_01 to SNP_18, especially at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 consecutive SNP markers selected from SNP_01 to SNP_09, with preferably at least SNP_03 comprising a Cytosine at nucleotide 101 of SEQ IDNO: 3 and optionally also one or more of SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_07, SNP_02 to SNP_07, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02 to SNP_05, or SNP_01 to SNP_04 or SNP_02 to SNP_04 comprising the donor SNP nucleotide.
- QTL11 (or a variant) is located on a sub-fragment comprising the donor haplotype of consecutive markers SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_07, SNP_02 to SNP_07, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02 to SNP_05, SNP_01 to SNP_04 or SNP_02 to SNP_04.
- the donor haplotype is as described herein above and in Table 1.
- the smaller sub- fragment retains QTL11 (or a variant thereof), i.e.
- the smaller fragment confers an increase resistance against A. gossypii (reduced susceptibility to A. gossypii), e.g. as described for the whole introgression fragment.
- Smaller fragments can be introgressed de novo from a wild Capsicum donor or can be generated from the plants grown from the deposited seeds, whereby new recombinant chromosomes 11 are generated.
- the introgression fragment on chromosome 11 comprising QTL11 is obtainable by crossing a plant grown from NCIMB43605 (or ancestors thereof or descendent/progeny therefrom) with another domesticated and cultivated Capsicum plant, especially a cultivated C. annuum plant.
- the cultivated Capsicum plant comprising QTL 11 may be a cultivated plant of a domesticated Capsicum species, especially a cultivated plant of the species C. annuum, C. baccatum, C. frutescens, C. chinense or C. pubescens.
- SNP markers which are indicative of the presence of the introgression fragment on chromosome 11
- the donor SNP nucleotide or genotype or SNP haplotype, when referring to several markers
- SNP nucleotide or genotype (and haplotype) as e.g.
- SNP marker genotype can distinguish between the introgression fragment being in homozygous or heterozygous form, as shown in the Table. In homozygous form the nucleotide is identical, while in heterozygous form the nucleotide is not identical.
- the SNP genotype of the ‘wild type’ chromosome lacking the introgression fragment is the other genotype, also listed in Table 1 (under genotype of recurrent parent) or may be any nucleotide for different recurrent parents. So, e.g.
- the genotype of SNP_01 indicative of the introgression fragment comprising QTL11 is ‘GG’ ( QTL11 / QTL11) or ‘GT’ or ‘GX’ (QTL11/ wt) while the SNP genotype indicative of the wild type / genetic control / control (lacking the introgression fragment) is ‘TT’ or ‘XX’ (wt/wt), with X being any nucleotide.
- a cultivated Capsicum plant especially a cultivated C. annuum plant
- an introgression fragment on chromosome 11 in homozygous or heterozygous form i.e. comprising QTL11 from a wild donor
- said introgression fragment (or sub-fragment) confers an increase in A. gossypii resistance (also referred to as a reduction in susceptibility) compared to the control plant lacking the introgression fragment on chromosome 11, e.g. the genetic control or control line or variety of preferably the same Capsicum type, when grown under the same conditions.
- the increase in A. gossypii resistance is phenotypically expressed as e.g. a (statistically) significantly lower average number of nymphs and/or adults being counted on the cultivated plant line or variety comprising the introgression fragment on chromosome 11 (preferably in homozygous form), compared to the control line or variety lacking the introgression fragment on chromosome 11 (e.g. the genetic control) when tested in e.g. a non-choice test (such as a clip-on cage assay) as described herein.
- aphid reproduction and/or survival is reduced due to QTL11 (or a variant thereof).
- gossypii resistance (or reduced susceptibility to A. gossypii) is phenotypically expressed as e.g. a (statistically) significantly reduced aphid population growth on the cultivated plant line or variety comprising the introgression fragment on chromosome 11 (preferably in homozygous form), compared to the control line or variety lacking the introgression fragment on chromosome 11 (e.g. the genetic control) when tested in e.g. a free-choice test (such as a population growth assay), as described herein.
- a free-choice test such as a population growth assay
- annuum plants and plant parts, especially sweet pepper plants, are provided in which the presence of QTL 11 (or a variant) in homozygous form results in the average number of new nymphs being (in e.g. a non-choice test such as a clip-on cage assay) equal to or less than 90% of the average number of nymphs on the control, preferably equal to or less than 80% of the control, equal to or less than 70% of the control, equal to or less than 60% of the control, equal to or less than 50% of the control or equal to or less than 40% of the control.
- a non-choice test such as a clip-on cage assay
- the average number of nymphs is reduced by at least 10%, 20%, 30%, 40%, 50% or 60% compared to the average number of nymphs on the control.
- the elite line comprising QTL 11 in homozygous form shows a reduction by 51.88% in average number of nymphs (the control comprises 61.4 nymphs and the elite line with QTL 11 comprises only 29.54 nymphs).
- the phenotypic effect of the QTL may be different, as different genetic backgrounds may have varying degrees of susceptibility to A. gossypii, but the effect of the QTL should still be measurable in an assay, such as a clip-on cage assay.
- an assay such as a clip-on cage assay.
- the controls should preferably be of the same Capsicum type, but lacking QTL 11 (or a variant thereof), and they are ideally genetically similar to the plant line with the QTL11 (or a variant thereof).
- the control may be the recurrent parent line or a genetic control. In this way the effect of the QTL on the reduction of susceptibility can be measured in any Capsicum line or type.
- the plants of the invention therefore, comprise a genome of cultivated Capsicum, with at least one or two recombinant chromosomes 11 (i.e. heterozygous or homozygous).
- the recombinant chromosomes comprise a fragment of a wild donor, which is easily distinguishable from the cultivated genome by molecular marker analysis (of e.g. one or more of the SNP markers linked to the QTL), whole genome sequencing, chromosome painting and similar techniques.
- the introgression fragment on chromosome 11 is from a wild donor, comprises the A. gossypii resistance conferring QTL, QTL 11 or a variant thereof, and comprises all or part of the region (i.e. a sub- region or sub-fragment) starting at SNP_01 (at nucleotide 101 in SEQ ID NO: 1) and ending at SNP_18 (at nucleotide 101 of SEQ ID NO: 18) of the chromosome, whereby a part of the region refers to a subregion or sub-fragment retaining QTL 11 as described above.
- the introgression fragment (or sub-fragment) comprises the QTL11 or a variant thereof and one or more or all (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) SNP markers of the wild donor selected from SNP_01 to SNP_18 (as shown in Table 1) or selected from the markers of a subregion as described above.
- the presence of the introgression fragment on chromosomes 11 in the genome of the plant or plant cell or plant tissue (or in the DNA extracted therefrom) is detectable by a molecular marker assay which detects one or more molecular markers of the introgression fragment (or sub-fragment or sub-region).
- a molecular marker assay which detects one or more molecular markers of the introgression fragment (or sub-fragment or sub-region).
- the SNP genotype of the markers may also be determined by sequencing or by using alternative markers located in between the SNP markers provided herein or within 7cM, or within 5cM, of a marker provided herein; or within 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4Mb, 0.3Mb, 0.2Mb, 0.1 Mb, 50kb, 20kb, lOkb, 5kb, 2kb, lkb or less of a marker provided herein.
- Cultivated Capsicum plants comprisins an introgression fragment on chromosome 11 (comprisins QTLll or a variant thereof)
- QTL11 or a variant of QTL11 is in one aspect located in the region between SNP 01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof) and SNP_18 at nucleotide 101 of SEQ ID NO: 18 (or a variant thereof).
- a cultivated Capsicum plant comprising an introgression fragment on chromosome 11 in homozygous or heterozygous form, wherein said introgression fragment confers an increase in resistance against infestation by the aphid A. gossypii (compared to the plant lacking the introgression fragment, e.g. the genetic control) and wherein said introgression fragment is detectable by a molecular marker assay (i.e.
- the plant comprises the donor nucleotide for one or more molecular markers) which detects at least 1, preferably at least 2 or 3, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the markers selected from the group consisting of: a) the GG (homozygous) or GT (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); b) the TT (homozygous) or TC (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); c) the CC (homozygous) or CA (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof
- SNP_12 at nucleotide 101 of SEQ ID NO: 12 (or in a variant thereof); m) the GG (homozygous) or GA (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_13 at nucleotide 101 of SEQ ID NO: 13 (or in a variant thereof); n) the TT (homozygous) or TC (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_14 at nucleotide 101 of SEQ ID NO: 14 (or in a variant thereof); o) the TT (homozygous) or TG (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_15 at nucleotide 101 of SEQ ID NO: 15 (or in a variant thereof); p) the AA (homozygous) or AG (heterozygous) genotype for the Single Nucleotide Poly
- the one or more markers include at least the marker under c) (for SNP_03), and optionally also under b) (for SNP_02) and/or d) (for SNP_04).
- further markers may be included such as under a) (for SNP_01) and/or e) (for SNP_05).
- optionally yet further markers may be included, under f) (for SNP_06), under g (for SNP_07), under h (for SNP_08) and/or under i (for SNP_09).
- variant sequence comprises at least 95% sequence identity with the mentioned sequence.
- the introgression fragment comprises the donor SNP haplotype or genotype of at least 3, 4, 5 or 6 of the markers listed under a) to r) above, or listed under a) to i) above, and comprises the QTL11 or a variant thereof.
- the introgression fragment is obtainable from (has as source) seeds deposited under NCIMB 43605 or progeny thereof, i.e. material derived from the deposit by selfing and/or crossing and retaining QTL11.
- the introgression fragment is obtainable from another wild Capsicum donor, comprising the same donor SNP haplotype for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of SNP_01 to SNP_18 (as shown in Table 1) or for 1, 2, 3, 4, 5, 6 or more markers of any of the sub-regions or sub-fragments described above.
- SNP_03 the marker under c
- the introgression fragment comprises the donor SNP haplotype or genotype of at least 6, 7, 8, 9 or 10 of the markers listed under a) to r) above, or listed under a) to i) above, and comprises the QTL11 or a variant thereof.
- the introgression fragment is obtainable from (has as source) seeds deposited under NCIMB43605 or progeny thereof.
- the introgression fragment is obtainable from another wild Capsicum donor, comprising the same donor SNP haplotype for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of SNP_01 to SNP_18 (as shown in Table 1) or for 1, 2, 3, 4, 5, 6 or more markers of any of the sub- regions or sub-fragments described above.
- at least the marker under c) (SNP_03) is present.
- the introgression fragment comprises the donor haplotype or genotype of at least 3, 4, 5 or 6 consecutive markers of the markers listed under a) to r) above, or listed under a) to i) above, and comprises the QTL11 or a variant thereof.
- the introgression fragment is obtainable from (has as source) seeds deposited under NCIMB43605 or progeny thereof.
- the introgression fragment is obtainable from another wild Capsicum donor, comprising the same donor SNP haplotype for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of SNP_01 to SNP_18 (as shown in Table 1) or for 1, 2, 3, 4, 5, 6 or more markers of any of the sub-regions or sub-fragments described above.
- the introgression fragment comprises the donor SNP haplotype or genotype of at least 6, 7, 8, 9 or 10 consecutive markers of the markers listed under a) to r) above, or listed under a) to i) above, and comprises the QTL11 or a variant thereof.
- the introgression fragment is obtainable from (has as source) seeds deposited under NCIMB43605 or progeny thereof.
- the introgression fragment is obtainable from another wild Capsicum donor, comprising the same donor SNP haplotype for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of SNP_01 to SNP_18 (as shown in Table 1) or for 1, 2, 3, 4, 5, 6 or more markers of any of the sub-regions or sub-fragments described above.
- SNP_03 the marker under c
- the introgression fragment comprises the donor SNP haplotype of at least SNP markers SNP_03, optionally also for SNP_02 and/or SNP_04.
- the skilled person can also develop other molecular markers, e.g. markers in-between marker SNP_01 and SNP_18 or in between SNP_01 and SNP_09 and/or within 7 cM or within 5 cM of any one of SNP_01 to SNP_18, and/or within 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4Mb, 0.3Mb, 0.2Mb, 0.1 Mb, 50kb, 20kb, lOkb, 5kb or less of any one of SNP_01 to SNP_18 or of any one of SNP_01 to SNP_09.
- markers may also be a stretch of nucleotide, CAPS markers, INDELs, etc.
- the skilled person can, for example, sequence the introgression fragment found in seeds deposited under accession number NCIMB43605 and use the sequence information to develop new markers and marker assays.
- Fine mapping has been carried out and identified the subregion flanked by SNP_01 and SNP_09 to comprise QTL 11. Further fine mapping can be carried out to identify which sub-region of the introgression fragment the QTL11 (or a variant thereof) is located on.
- QTL11 (or a variant thereof) is located in the region comprising SNP_01 to SNP_9, or SNP_02 to SNP_09, or SNP_01 to SNP_08, or SNP_02 to SNP_08, or SNP_01 to SNP_07 or SNP_02 to SNP_07, or SNP_01 to SNP_06 or SNP_02 to SNP_06, or SNP_01 to SNP_05 or SNP_02 to SNP_05, or SNP_01 to SNP_04 or SNP_02 to SNP_04.
- a cultivated Capsicum plant comprising an introgression fragment on chromosome 11 in homozygous or heterozygous form, wherein said introgression fragment confers an increase in resistance against A. gossypii (compared to the plant lacking the introgression fragment, e.g.
- said introgression fragment comprises the following markers: the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); - the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 09 at nucleotide 101 of SEQ ID NO: 9 (or in a variant thereof); or the following markers: the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); - the AA (homozygous) or AG or AX (heterozygous) genotyp
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 09 at nucleotide 101 of SEQ ID NO: 9 (or in a variant thereof); or the following markers: the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); - the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotyp
- Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); - the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucle
- Polymorphism marker SNP_08 at nucleotide 101 of SEQ ID NO: 8 (or in a variant thereof); or the following markers: the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); - the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); - the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP_08 at nucleotide 101 of SEQ ID NO: 8 (or in a variant thereof) or the following markers: the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); - the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous)
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); or the following markers: the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for
- Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); or the following markers: - the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotyp
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); or the following markers: the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); - the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); or the following markers: the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); - the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotyp
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); or the following markers: the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); - the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous)
- the introgression fragment comprising the QTL may, thus, be large (comprising the donor haplotype for SNP_01 to SNP_18), or may be smaller and lack markers, but it may still confer A. gossypii resistance on the cultivated Capsicum plant, i.e. it can still comprise the QTL (QTL11 or a variant).
- Such smaller introgression fragments are an embodiment of the invention. Plants having smaller introgression fragments (e.g. sub- fragments) which still confer the enhanced A. gossypii resistance (i.e. contain the QTL11 or a variant) can be generated using known techniques, such as further fine-mapping or similar techniques.
- Marker assays can be used to select recombinants and to determine the size of the smaller introgression fragment.
- One or more of SNP markers of the donor genotype or haplotype may be missing.
- the cultivated Capsicum genotype (or haplotype) is then detected for these SNP markers.
- the A. gossypii resistance of plants comprising such a smaller introgression fragment can then be compared in a resistance assay as described herein, e.g. a non-choice assay and/or a free-choice assay. For example, if in a clip-on cage assay the average number of nymphs and/or adults is significantly reduced compared to the control, then the smaller introgression fragment (or sub-fragment) has retained the QTL11.
- the same or variant QTL may be introgressed from a different wild Capsicum donor, whereby optionally not all SNP markers disclosed herein are present, i.e. the SNP haplotype may be slightly different than for the specific C. baccatum donor used herein.
- Such alternative wild Capsicum sources can be identified using the SNP markers provided herein, by screening germplasm (i.e. accessions of) wild Capsicum using a marker assay to detect the genotype or haplotype of markers SNP_01 to SNP_18, or of subgroups thereof, such as 6, 7, 8, 9, 10 consecutive markers or any subgroups described above. Plants comprising the same or variant QTL 11 from other sources are also an embodiment of the invention.
- the donor has a SNP haplotype which is identical to the specific C. baccatum donor described herein for at least 3, 4, 5, 6, 7, 8, 9, 10 or more (or all) of the SNPs of SNP_01 to SNP_18 (especially for at least 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive SNP markers of SNP_01 to SNP_18) or for at least 1, 2, 3, 4, 5, or more (or all) of SNP_01 to SNP_09 or any subgroup thereof (e.g. SNP_02 to SNP_09, SNP_01 to SNP_08, etc.) and preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04, the donor may comprise QTL11.
- the skilled person can then introgress the QTL11 (or a variant thereof) into cultivated Capsicum in order to enhance A. gossypii resistance as described herein and in order to confirm that the QTL reduces susceptibility to A. gossypii when present in cultivated Capsicum.
- Wild Capsicum donors may be accessions of wild Capsicum annuum, wild C. baccatum, wild C. frutescens, wild C. chinense, wild C. pubescens, C. galapagoense, C. chacoense, C. tovarii, C. eximium, C. cardenasii, or C. praetermissum.
- Such wild accessions are for example available from seedbanks, such as the CGN collection of Wageningen University or the GRIN collection in the USA, the U.S. National Plant Germplasm System found on the world wide web at npgsweb.ars-grin.gov/gringlobal/search.aspx.
- 67 Capsicum accessions having the status ‘wild’ can be found.
- the wild donor is a wild C. baccatum, optionally a wild C. baccatum var. baccatum, a wild C. baccatum var. pendulum, or a wild C. baccatum var. praetermissum.
- the QTL11 or variant QTL11 is preferably introgressed into cultivated Capsicum, such as any cultivated plant of a domesticated species, selected from cultivated C. annuum, cultivated C. baccatum, cultivated C. frutescens, cultivated C. chinense and cultivated C. pubescens. Introgression may need the use of bridge species and/or embryo rescue techniques, as known from crossability studies for Capsicum species (crossability polygons) published.
- the cultivated Capsicum plant of a domesticated species comprises an introgression fragment comprising QTL11 (or a variant) and at least a subset of SNP markers with the genotype or haplotype of the wild Capsicum donor, i.e. for at least 3, 4, 5, 6, 7, 8, 9 or 10 (or more) SNP markers of SNP_01 to SNP_18, especially for at least 3, 4, 5, 6, 7, 8, 9. 10 or more consecutive SNP markers of SNP_01 to SNP_18, preferably including at least SNP_03 and optionally SNP_02 and/or SNP_04.
- sub-fragments are fragments which have the same SNP haplotype for one of the following groups of SNP markers as the C.
- baccatum donor described herein see also Table 1: SNP_01 to SNP_18, SNP_02 to SNP_17, SNP_02 to SNP_15, SNP_02 to SNP_12, SNP_02 to SNP_10, SNP_03 to SNP_12, SNP_03 to
- SNP_10 or SNP_01 to SNP_06. Or preferably SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08,
- SNP_02 to SNP_08 SNP_01 to SNP_07, SNP_02 to SNP_07, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02 to SNP_05, SNP_01 to SNP_04, SNP_02 to SNP_04, SNP_03 and optionally SNP_02 and/or SNP_04.
- the cultivated Capsicum plant comprises the same SNP haplotype for all, or all except 1 or 2 or 3 markers of SNP_01 to SNP_18, SNP_02 to SNP_17, SNP_02 to SNP_15 , SNP_02 to SNP_12, SNP_03 to SNP_12, SNP_03 to SNP_10, or SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_07, SNP_02 to SNP_07, SNP_01 to SNP_06, SNP_02 to
- SNP_06 SNP_01 to SNP_05, SNP_02 to SNP_05, SNP_01 to SNP_04, SNP_02 to SNP_04.
- the introgression fragment (and a cultivated Capsicum plant or plant part, e.g., a cell, comprising the introgression fragment) can be detected in a marker assay by detecting the SNP genotype or haplotype of the introgression fragment (i.e. of the wild donor) of one or more or all of the markers above.
- a Quantitative Trait Locus (QTL11 or a variant thereof) was found to be present on chromosome 11 of a wild Capsicum donor which, when transferred (introgressed) into a cultivated Capsicum plant, variety or breeding line, and, when present in homozygous form or heterozygous form, confers significantly enhanced resistance (reduced susceptibility) to A. gossypii onto the cultivated plant.
- the QTL (or variant QTL) can be identified in wild donors and can then be introgressed into cultivated Capsicum, e.g. using MAS, i.e. using one or more (or all) of the SNP markers provided herein to detect and/or select progeny plants (e.g. backcross plants) comprising a recombinant chromosome 11.
- the selected plants i.e.
- the cultivated Capsicum plants comprising an introgression fragment on chromosome 11, wherein the introgression fragment on chromosome 11 is detectable by the donor SNP haplotype or genotype of one or more of the SNP markers SNP_01 to SNP_18, one or more of SNP_02 to SNP_17, one or more of SNP_02 to SNP_15, one or more of SNP_02 to SNP_12,one or more of SNP_02 to SNP_10, one or more of SNP_03 to SNP_12, one or more of SNP_03 to SNP_10, one or more of SNP_01 to SNP_06, preferably one or more of SNP_01 to SNP_09, one or more of SNP_02 to SNP_09, one or more of SNP_01 to SNP_08, one or more of SNP_02 to SNP_08, one or more of SNP_01 to SNP_07, one or more of SNP_02 to SNP_07, one or more of S
- Accessions of wild Capsicum are obtainable from the USDA National Plant Germplasm System collection or other seed collections, and can thus be screened for the presence of QTL 11 using e.g. a marker assay as described herein, and accessions comprising one or more of the SNP haplotype or genotype of the donor can be crossed with a cultivated Capsicum plant having normal wild-type, non-recombinant chromosomes 11.
- the F 1 or F2 generation (or further generation, such as the F3 or a backcross generation) can then be screened for recombinant plants having the introgression fragment using molecular marker assays described herein, for detecting one or more of the SNP markers.
- the introgression fragment comprising the QTL 11 is derivable from (or derived from) or obtainable from (or obtained from; or as present in) seeds, a representative sample of which has been deposited under accession number NCIMB 43605, or from progeny thereof.
- the progeny may be any progeny which retain the one or more (or all) SNP markers indicative of (and linked to) the QTL, as described.
- progeny are not limited to F 1 or F2 progeny of the deposit, but can be any progeny, whether obtained by selfing and/or crossing with another Capsicum plant, e.g. another cultivated C. annuum line.
- the introgression fragment is identifiable by one or more of the markers described elsewhere herein, especially markers SNP_01 to SNP_18 for the introgression fragment on chromosome 11, or a subset of markers, such as one or more of the markers of SNP_01 to SNP_09 (or any other sub-region described).
- the invention provides a cultivated Capsicum plant of a domesticated species of Capsicum (especially C. annuum), having a genome of cultivated Capsicum which comprises increased resistance against A.
- the increased resistance is conferred by an introgression fragment on the cultivated Capsicum chromosome 11, wherein said introgression fragment is obtained by (or obtainable by) crossing a cultivated plant grown from seeds deposited under NCIMB 43605 or progeny of this plant (which comprises one or more the markers disclosed herein linked to the QTL) with a cultivated Capsicum plant, especially a cultivated C. annuum plant.
- a cultivated Capsicum plant especially the cultivated C.
- annuum plant comprises the same introgression fragment and the same recombinant chromosome 11 as present in NCIMB 43605 (comprising the wild donor genotype or haplotype for SNP_01 to SNP_18), or it comprises a shorter fragment of that introgression fragment (a sub-fragment or sub-region), whereby the shorter fragment retains the genetic element conferring enhanced resistance against A. gossypii (QTL11).
- the shorter sub-fragment comprises at least the donor SNP nucleotide for SNP_03, optionally also for SNP_02 and/or SNP_04.
- the invention relates to a plant of the invention i.e. a cultivated Capsicum (especially a cultivated C. annuum) plant comprising an introgression fragment from a wild Capsicum plant on chromosome 11 in homozygous or heterozygous form and wherein said introgression fragment is the introgression fragment “as in” / is “identical to” / is “the same as in” the seeds deposited under number NCIMB 43605, or is a shorter fragment (sub fragment or sub region) thereof, but still confers enhanced A. gossypii resistance due to the presence of QTL11 on the sub-fragment.
- a plant of the invention i.e. a cultivated Capsicum (especially a cultivated C. annuum) plant comprising an introgression fragment from a wild Capsicum plant on chromosome 11 in homozygous or heterozygous form and wherein said introgression fragment is the introgression fragment “as in” / is “identical to”
- the invention relates to a plant of the invention i.e. a cultivated Capsicum (especially a cultivated C. annuum) plant comprising an introgression fragment from a wild donor on chromosome 11, in homozygous or heterozygous form, and wherein said introgression fragment is a variant of the introgression fragment found in seeds deposited under number NCIMB 43605, i.e. it comprises the QTL 11, but the genomic sequence may be different.
- a plant of the invention i.e. a cultivated Capsicum (especially a cultivated C. annuum) plant comprising an introgression fragment from a wild donor on chromosome 11, in homozygous or heterozygous form, and wherein said introgression fragment is a variant of the introgression fragment found in seeds deposited under number NCIMB 43605, i.e. it comprises the QTL 11, but the genomic sequence may be different.
- the genomic sequence of an introgression fragment comprising QTL11 from other wild donors will most likely not be identical to the genomic sequence as introgressed into NCIMB 43605, and even the resistance conferring gene (comprising a promoter, introns and exons) may be divergent in nucleotide sequence, but the function will be the same, i.e. conferring enhanced A. gossypii resistance.
- the divergence can be seen in that the SNP donor haplotype of certain SNP markers linked to QTL 11 may be not 100% identical to the donor haplotype found in NCIMB 43605.
- not all of SNP_01 to SNP_18, especially not all of SNP_01 to SNP_09 (or not all markers of any other subregion described) may have the same donor haplotype in other wild donors.
- other donors may comprise the same donor SNP haplotype as in NCIMB43605 for e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more SNP markers selected from SNP_01 to SNP_18, or e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more consecutive SNP markers selected from SNP_01 to SNP_18, or e.g.
- SNP markers selected from SNP_01 to SNP_18, SNP_02 to SNP_17, SNP_02 to SNP_15, SNP_02 to SNP_12, SNP_03 to SNP_12, SNP_03 to SNP_10, SNP_01 to SNP_06, SNP_03 to SNP_10 or or SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_07, SNP_02 to SNP_07, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02 to SNP_05, SNP_01 to SNP_04, or SNP_02 to SNP_04, or for at least SNP_03 and optionally SNP_02 and/or SNP_04.
- the A. gossypii resistance QTL, QTL11 (comprising e.g. a variant or ortholog of the A. gossypii resistance allele) may still be present in such wild accessions.
- the skilled person is capable of identifying and introgressing the QTL 11 comprising region found in other wild Capsicum accessions into cultivated Capsicum, e.g. identifying wild Capsicum accessions comprising the (donor) SNP markers or a subset thereof and transferring these SNP markers (or subset) into a cultivated Capsicum plant line or variety and assessing the A. gossypii resistance of the cultivated plant compared to the control plant lacking the SNP markers (or subset), i.e. lacking the introgression fragment.
- a plant or plant part comprising QTL 11 or a variant thereof wherein the introgression fragment in the plant or plant part comprising QTL 11, comprises and/or is detectable by a molecular marker assay which detects the following genotypes or haplotypes for at least 1, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 Single Nucleotide Polymorphism (SNP) markers selected from the group consisting of: a) the GG (homozygous) or GX (heterozygous) genotype for SNP_01 at nucleotide 101 of SEQ ID NO:
- SNP Single Nucleotide Polymorphism
- the one or more markers include at least the marker under c) (for SNP_03), and optionally also under b) (for SNP_02) and/or d) (for SNP_04).
- further markers may be included such as under a) (for SNP_01) and/or e) (for SNP_05).
- optionally yet further markers may be included, under f) (for SNP_06), under g (for SNP_07), under h (for SNP_08) and/or under i (for SNP_09).
- a plant or plant part comprising QTL11 or a variant thereof comprises and/or is detectable by a molecular marker assay which detects the following genotypes or haplotypes for at least 1, preferably at least 2, 3, 4, 5, 6, 7, 8, or 9 Single Nucleotide Polymorphism (SNP) markers selected from the group consisting of: a) the GG (homozygous) or GX (heterozygous) genotype for SNP_01 at nucleotide 101 of SEQ ID NO:
- SNP Single Nucleotide Polymorphism
- the one or more markers include at least the marker under c) (for SNP_03), and optionally also under b) (for SNP_02) and/or d) (for SNP_04).
- further markers may be included such as under a) (for SNP_01) and/or e) (for SNP_05).
- optionally yet further markers may be included, under f) (for SNP_06), under g (for SNP_07), under h (for SNP_08) and/or under i (for SNP_09).
- the first nucleotide mentioned above is the nucleotide of the donor (of the introgression fragment, which is part of the recombinant chromosome 11), while the second nucleotide is the nucleotide of the chromosome 11 lacking the introgression fragment, e.g. the non-recombinant chromosome 11 of the recurrent parent.
- the second nucleotide is herein indicated as X, meaning any nucleotide (A, G, T or C), as this second nucleotide can vary, depending on the genetic background of the Capsicum line.
- nucleotide X is the nucleotide of the recurrent parent as present in the deposited seeds and as indicated in Table 1 and herein below.
- a plant or plant part comprising QTL11 or a variant thereof comprises and/or is detectable by a molecular marker assay which detects the following genotypes or haplotypes for at least 1, 2, 3, preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 Single Nucleotide Polymorphism (SNP) markers selected from the group consisting of: a) the GG (homozygous) or GT (heterozygous) genotype for SNP_01 at nucleotide 101 of SEQ ID NO:
- SNP Single Nucleotide Polymorphism
- the one or more markers include at least the marker under c) (for SNP_03), and optionally also under b) (for SNP_02) and/or d) (for SNP_04).
- further markers may be included such as under a) (for SNP_01) and/or e) (for SNP_05).
- optionally yet further markers may be included, under f) (for SNP_06), under g (for SNP_07), under h (for SNP_08) and/or under i (for SNP_09).
- a plant or plant part comprising QTL 11 or a variant thereof wherein the introgression fragment in the plant or plant part comprising QTL 11, comprises and/or is detectable by a molecular marker assay which detects the following genotypes or haplotypes for at least 1, 2, 3, preferably at least 4, 5, 6, 7, 8 or 9 Single Nucleotide Polymorphism (SNP) markers selected from the group consisting of: a) the GG (homozygous) or GT (heterozygous) genotype for SNP_01 at nucleotide 101 of SEQ ID NO:
- SNP Single Nucleotide Polymorphism
- the one or more markers include at least the marker under c) (for SNP_03), and optionally also under b) (for SNP_02) and/or d) (for SNP_04).
- further markers may be included such as under a) (for SNP_01) and/or e) (for SNP_05).
- optionally yet further markers may be included, under f) (for SNP_06), under g (for SNP_07), under h (for SNP_08) and/or under i (for SNP_09).
- the genotype or haplotype of at least 1, 2, 3, or at least 4, 5, 6, 7, 8, 9 or more markers which are detected (and which are present on the introgression fragment) are consecutive markers. That means the introgression fragment comprises the genotype (homozygous introgression fragment) or haplotype (heterozygous introgression fragment) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or more markers, whereby these are consecutive markers, e.g. three consecutive markers are for example SNP_01, SNP_02, SNP_03 or alternatively SNP_14, SNP_15 and SNP_16.
- the genotype of the plant or plant part comprising the introgression fragment in homozygous form would then be GG-TT-CC or TT-TT-AA, respectively.
- the marker haplotype of any 7 or more consecutive markers out of the 18 markers is a unique identifier of the introgression fragment of the donor
- the marker haplotype comprising at least the donor SNP haplotype for SNP_04, SNP_11 and/or SNP_15 is a unique identifier of the introgression fragment of the donor.
- haplotype is indicted to be the haplotype of a specific SNP nucleotide in a specific sequence (SEQ ID Numbers), or in a sequence comprising at least 95% sequence identity (or 96%, 95%, 98% or 99%) to the specific sequence (SEQ ID Number).
- SEQ ID Numbers specific sequence
- GG homozygous
- GT heterozygous genotype for SNP_01 at nucleotide 101 of SEQ ID NO: 1 “or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1”.
- the sequence of SEQ ID Number flanking the SNP i.e.
- flanking sequences which comprise e.g. one, two or three nucleotides which are different from the flanking sequences provided herein in the specific SEQ ID Numbers are encompassed herein.
- the SNP haplotype of one or more or all SNP markers linked to QTL11 is useful for the genetic identification of the donor fragment and/or introgression fragment in combination with the phenotype conferred by the QTL, and thus for the identification of wild donors comprising the QTL 11 (or a variant thereof), for generating cultivated Capsicum plants or plant parts comprising the QTL11 (or a variant thereof) and/or detecting wild or cultivated Capsicum plants or plant parts comprising the QTL11 (or a variant thereof).
- a method of using the SNP haplotype of one or more or all SNP markers linked to QTL11 (or a variant thereof) for breeding Capsicum plants comprising the QTL11 (or a variant thereof) and/or for screening donor accessions or cultivated Capsicum lines or varieties for the presence of QTL11 is one embodiment herein.
- a method for detecting, selecting and/or breeding a plant or plant part comprising QTL11 or a variant thereof comprising carrying out a molecular marker assay and optionally selecting a plant or plant part which comprises the following genotypes or haplotypes for at least 1, 2, 3, preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 Single Nucleotide Polymorphism (SNP) markers selected from the group consisting of: a) the GG (homozygous) or GX or GT (heterozygous) genotype for SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1), b) the TT (homozygous) or TX or TC (heterozygous) genotype for SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence
- SNP Single Nucle
- the at least 1, 2, 3 markers include at least the marker under c) (for SNP_03), and optionally also under b) (for SNP_02) and/or d) (for SNP_04).
- further markers may be included such as under a) (for SNP_01) and/or e) (for SNP_05).
- markers may be included, under f) (for SNP_06), under g (for SNP_07), under h (for SNP_08) and/or under i (for SNP_09).
- a method for detecting, selecting and/or breeding a plant or plant part comprising QTL 11 or a variant thereof comprising carrying out a molecular marker assay and optionally selecting a plant or plant part which comprises the following genotypes or haplotypes for at least 1, 2, 3, preferably at least 4, 5, 6, 7, 8 or 9 Single Nucleotide Polymorphism (SNP) markers selected from the group consisting of: a) the GG (homozygous) or GX or GT (heterozygous) genotype for SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or at nucleotide 101 of a sequence comprising at least 95% identity to SEQ ID NO: 1), b) the TT (homozygous) or TX or TC (heterozygous) genotype for SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or at nucleotide 101 of a sequence comprising at least 95% identity to SNP
- SNP Single Nu
- the at least 1, 2, 3 or more markers include at least the marker under c) (for SNP_03), and optionally also under b) (for SNP_02) and/or d) (for SNP_04).
- further markers may be included such as under a) (for SNP_01) and/or e) (for SNP_05).
- markers may be included, under f) (for SNP_06), under g (for SNP_07), under h (for SNP_08) and/or under i (for SNP_09).
- the plant or plant parts may be of one or more wild donor accessions, or of one or more breeding lines or varieties.
- X may be any nucleotide.
- the selected plant or plants may be phenotyped for resistance against A. gossypii.
- a method for detecting and/or selecting and/or breeding a plant or plant part comprising QTL11 or a variant thereof comprising carrying out a molecular marker assay and optionally selecting a plant or plant part which comprises the following genotypes or haplotypes for at least 1, 2, 3, preferably at least 4, 5, 6, 7, 8 or 9 Single Nucleotide Polymorphism (SNP) markers selected from the group consisting of: the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or C
- Polymorphism marker SNP_08 at nucleotide 101 of SEQ ID NO: 8 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_09 at nucleotide 101 of SEQ ID NO: 9 (or in a variant thereof); or selected from the group consisting of: the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozyg
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); - the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_08 at nucleotide 101 of SEQ ID NO: 8 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 09 at nucleotide 101 of SEQ ID NO: 9 (or in a variant thereof); or selected from the group consisting of: the GG (homozygous) or GT or GX (heterozygous) genotyp
- Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleot
- Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); - the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP_08 at nucleotide 101 of SEQ ID NO: 8 (or in a variant thereof) or selected from the group consisting of: - the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (hetero
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); - the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide
- Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); or selected from the group consisting of: - the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozy
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); the CC (homozygous) or CT or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_07 at nucleotide 101 of SEQ ID NO: 7 (or in a variant thereof); or selected from the group consisting of: the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); or selected from the group consisting of: - the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozy
- Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); the GG (homozygous) or GA or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP 06 at nucleotide 101 of SEQ ID NO: 6 (or in a variant thereof); or selected from the group consisting of: - the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozy
- Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_05 at nucleotide 101 of SEQ ID NO: 5 (or in a variant thereof); or selected from the group consisting of: the GG (homozygous) or GT or GX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_01 at nucleotide 101 of SEQ ID NO: 1 (or in a variant thereof); the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); the CC (homozygous) or CA or CX (heterozyg
- Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof); or selected from the group consisting of: the TT (homozygous) or TC or TX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_02 at nucleotide 101 of SEQ ID NO: 2 (or in a variant thereof); - the CC (homozygous) or CA or CX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_03 at nucleotide 101 of SEQ ID NO: 3 (or in a variant thereof); the AA (homozygous) or AG or AX (heterozygous) genotype for the Single Nucleotide Polymorphism marker SNP_04 at nucleotide 101 of SEQ ID NO: 4 (or in a variant thereof) or selected from the group consisting of: - the TT (homozyg
- the selected plant or plants may be phenotyped for resistance against A. gossypii.
- the plant or plant part comprising it in the plant genome (on chromosome 11) is preferably selected.
- the plant or plant part may be a wild Capsicum species (e.g. C. baccatum) or a domesticated and/or cultivated Capsicum species (e.g. C. annuum).
- the selected plant comprising the QTL11 will show the increase in resistance against A. gossypii conferred by the QTL.
- the SNP genotype refers to two nucleotides, and genomic sequences comprising one of these two nucleotides, one on each chromosome 11. So a plant having a GG genotype for SNP 01 has an identical nucleotide (G) on both chromosomes (i.e. is homozygous for the introgression fragment), while a plant having e.g.
- an GT genotype for SNP 01 has one chromosome with an G at nucleotide 101 of SEQ ID NO: 1 (or at the equivalent nucleotide of a genomic sequence comprising at least 95% sequence identity to SEQ ID NO: 1) and one chromosome with a T at nucleotide 101 of SEQ ID NO: 1 and is heterozygous for the introgression fragment.
- the nucleotide for the chromosome lacking the introgression fragment may also be indicated as X, wherein X is selected from any nucleotide (A, T, G, C).
- the genotype for SNP_01, wherein the introgression fragment is in heterozygous form may thus be indicated as GX. The same applies to the other SNP markers.
- genomic sequences around the SNP markers provided herein may vary slightly from the reference genome sequence, e.g. in introgression fragments from this and other wild donors (i.e. variants or orthologous chromosome 11 regions) it is clear that the nucleotide sequences before and after the SNP may not be 100% identical to the sequences provided herein. Therefore sequences having at least 95% sequence identity to the sequences provided herein (when aligned over the entire length as defined), but which comprise the same SNP genotype, are encompassed herein.
- the introgression fragment, or the chromosome 11 region (or variant or orthologous chromosome 11 region) comprising the QTL (QTL 11 or variant), which is detectable by the above one or more markers is from a wild Capsicum, especially from wild C. baccatum, e.g. C. baccatum var. baccatum. In one aspect it is the same introgression fragment as found on chromosome 11 in seeds deposited under accession number NCIMB 43605, or a smaller fragment (sub-fragment) retaining the QTL.
- the introgression fragment on chromosome 11 is equal to or less than 60Mb in size, preferably equal to or less than 50Mb, 45Mb, 40Mb, 35Mb, 30Mb, 25Mb, 20Mb, 15Mb, 10Mb, 5Mb, 1 Mb in size, preferably equal to or less than 0.5 Mb in size.
- the introgression fragment is at least 0.1 Mb, 0.2 Mb, or 0.3 Mb, 0.5 Mb, 1Mb, 5Mb or 10Mb in size.
- various ranges of introgression fragment sizes are encompassed herein, such as fragments less than 20 Mb but more than 0.3 Mb which retain the QTL11 and the donor genotype or donor haplotype for one or more of the SNP markers (preferably consecutive markers) of SNP_01 to SNP_18, or for one or more of the SNP markers of a subgroup of SNP markers comprising QTL11, as described, preferably of one of the following subgroups: SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02 to SNP_05, SNP_01 to SNP_04, SNP_02 to SNP_04, SNP_02 to SNP_03 or SNP_03 to SNP_04.
- the size of an introgression fragment can be easily determined by e.g. whole genome sequencing or Next Generation Sequencing, e.g. as described in Qi el al. 2013 (supra) or in Huang el al. 2009 (supra).
- Especially introgression regions can be easily distinguished from cultivated genomic regions due to the larger amount of genetic variation (SNPs, INDELs, etc.) in the introgression region.
- NCIMB 43605 contains two recombinant chromosomes 11 (comprising the introgression fragment comprising QTL 11 in homozygous form) all of the F 1 seed and plants grown therefrom will contain one recombinant chromosome 11 from the NCIMB 43605 parent and one non recombinant chromosome 11 from the other cultivated parent.
- QTL11 can be transferred into any cultivated Capsicum breeding line or variety.
- QTL11 can be transferred into any cultivated Capsicum breeding line or variety.
- Progeny plants which comprise the QTL11 can be screened for, and selected for, by the presence of the donor genotype or haplotype for one or more of the above SNP markers.
- NCIMB 43605 To generate shorter introgression fragments, e.g. sub-fragments of the fragment present in NCIMB 43605, meiosis needs to take place and plants comprising the recombinant chromosomes 11, and especially new meiotic recombination events within the introgression fragment, need to be identified.
- seeds of NCIMB43605 can be selfed one or more times to produce FI, F2 or F3 plants (or further selfing generations), and/or FI, F2 or F3 plants (etc.) comprising the recombinant chromosome 11 can be backcrossed to a cultivated parent.
- Plants which comprise the recombinant chromosome 11 can be screened for, and selected for, by the presence of the donor genotype or haplotype of one or more of the above SNP markers (or subgroups thereof, as described) in order to identify plants comprising a smaller introgression fragment. Such new recombinants can then be tested for the presence of the QTL 11 on the smaller introgression fragment by determining the A. gossypii resistance compared to the (genetic) control lacking the introgression fragment, and optionally compared to the resistance of plants grown from seeds deposited under NCIMB43605.
- cultivated Capsicum plants comprising QTL 11 (or a variant thereof) can be generated and/or identified using different methods. For example, to obtain a cultivated Capsicum plant comprising a introgression fragment from a wild donor, first a wild donor is identified which comprises the donor SNP genotype or haplotype one or more of the SNP markers linked to QTL11 disclosed herein, e.g.
- the identified donor plant is then e.g. crossed with a cultivated Capsicum plant to obtain FI seeds.
- the FI can be selfed to produce F2, F3, etc.
- Plants which are comprising QTL11 can be screened for, and/or selected for, by the presence of the donor genotype or haplotype of one or more of the above SNP markers (or subgroups) and/or screened for, and/or selected for, an increased A. gossypii resistance phenotype compared to e.g. the initial cultivated parent (lacking the introgression).
- QTL mapping or sequencing can be carried out in order to identify further molecular markers linked to the QTL11 (or a variant thereof) and/or to generate cultivated Capsicum plants comprising an introgression fragment on chromosome 11 which confers an increased resistance against A. gossypii.
- the introgression fragment in a cultivated Capsicum plant or plant part, or a fragment / region in a wild Capsicum plant or plant part, comprising QTL11 (or a variant thereof), comprises: a) the donor SNP haplotype of at least 1, 2, 3, 4, 5, 6, 7, 8 or more SNP markers of SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02to SNP_05, SNP_01 to SNP_04, SNP_02 to SNP_04, SNP_02 to SNP_03 or SNP_03 to SNP_04; and preferably at least the donor SNP haplotype for SNP_03 and optionally for SNP_02 and/or SNP_04; b) the donor SNP haplotype of at least 2, 3, 4, 5, 6 or more
- different wild Capsicum accessions can be screened for the SNP haplotype of a), b) or c) above to identify other donor accessions than the one used herein (C. baccatum var baccatum ) which comprise QTL 11.
- the donor accessions can be screened also phenotypically for the susceptibility or resistance against A. gossypii.
- a wild Capsicum accession identified or selected for having the donor SNP haplotype of a), b) or c) can then be used to backcross QTL11 into cultivated Capsicum, e.g. into cultivated C. annuum.
- a cultivated Capsicum plant comprising an introgression fragment comprising QTL11 (or a variant thereof) and comprising the donor SNP haplotype of a), b) or c) above.
- the cultivated Capsicum is preferably tested phenotypically for the susceptibility or resistance against A. gossypii.
- the susceptibility should be significantly reduced in the cultivated Capsicum plant comprising QTL 11 (or a variant thereof).
- a plant cell, tissue or plant part of a plant or of a seed comprising at least one recombinant chromosome 11, wherein said recombinant chromosome 11 comprises an introgression fragment from a wild Capsicum and wherein said introgression fragment comprises QTL 11 (or a variant thereof) conferring significantly resistance against A. gossypii.
- a method for identifying a Capsicum plant comprising QTL 11 (or an introgression fragment comprising QTL 11, or a variant thereof, or in case a wild Capsicum is analyzed whether the wild genome contain a SNP haplotype indicating the presence of QTL11 or a variant thereof), comprising: a) screening a Capsicum plant using a molecular marker assay which detects the SNP marker genotype of the plant for one or more markers of SNP_01 to SNP_18, especially one or more markers of SNP_01 to SNP_09; b) optionally identifying and/or selecting a plant comprising the resistant donor SNP marker genotype for: i) the donor SNP haplotype of at least 1, 2, 3, 4, 5, 6, 7, 8 or more SNP markers of SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_06, SNP_02 to SNP_
- the Capsicum plant may be a cultivated Capsicum plant, such as a C. annuum plant, or a wild Capsicum plant, such as a wild C. baccatum.
- a population of Capsicum plants can be screened for the SNP marker genotype or haplotype.
- Such a population may e.g. several plants of cultivated Capsicum (e.g. C. annuum ) and/or several wild Capsicum accessions, in order to identify an accession which comprises QTL11 or a variant thereof.
- a method for identifying and/or selecting plant or plant part comprising an introgression fragment in a cultivated Capsicum (e.g. C. annuum ) plant or plant part, or a donor fragment in a wild Capsicum plant or plant part, comprising QTL11 (or a variant thereof), is provided comprising DNA analysis to identify and/or select a plant or plant part, wherein the fragment comprises: a) the donor SNP haplotype of at least 1, 2, 3, 4, 5, 6, 7, 8 or more SNP markers of SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02 to SNP_05, SNP_01 to SNP_04, SNP_02 to SNP_04, SNP_02 to SNP_03 or SNP_03 to SNP_04; and
- the DNA analysis in the above method can involve e.g. SNP genotyping, DNA sequencing, PCR analysis or other methods to determine the SNP haplotype of the plant or plant part.
- the molecular markers described herein may be detected according to standard method.
- SNP markers can easily be detected using a KASP-assay (see www.kpbioscience.co.uk) or other SNP genotyping assays.
- KASP-assay for example 50 base pairs upstream and 50 base pairs downstream of the SNP can be selected and two allele-specific forward primers and one allele specific reverse primer can be designed. See e.g. Allen et al. 2011, Plant Biotechnology J. 9, 1086-1099, especially p097-1098 for KASP assay method.
- the SNP markers and the presence/absence of the marker associated with the QTLs is determined using a KASP assay, but equally other SNP genotyping assays can be used.
- SNP genotyping assays can be used.
- HRM High Resolution Melting
- SNP- genotyping arrays e.g. Fluidigm, Illumina, etc.
- DNA sequencing may equally be used.
- the physical size of an introgression fragment can be determined by various methods, such as physical mapping, sequencing or by visualization of the introgression using Fluorescent in situ hybridization (FISH) images (Verlaan et al. 2011, Plant Journal 68: 1093-1103).
- FISH Fluorescent in situ hybridization
- the cultivated Capsicum plant especially a cultivated C. annuum plant, as described herein may be an inbred line, an OP (open pollinated variety) or an FI hybrid.
- the FI hybrid comprises only one recombinant chromosome 11 (comprising the introgression fragment with the QTL), i.e. the FI hybrid is heterozygous for the introgression fragment and the SNP markers described herein.
- Such an FI hybrid is produced by crossing two inbred parent lines, one of which possesses the introgression fragment (preferably in homozygous form, although not necessarily) and collecting the FI hybrid seeds from said cross.
- the FI hybrid may comprise the introgression fragment in homozygous form, i.e. produced by crossing two inbred parent lines, each comprising the introgression fragment in homozygous or heterozygous form.
- the cultivated Capsicum plant of the invention can be of any type such as blocky bell type, Cayenne type, Lamuyo, Dulce Italiano, Conical, Capia, Sweet Charleston, Dolma, Cherry, Jalapeno type, Shakira, Pencil or Hot Charleston, Sivri, Hungarian Wax, Kapya, banana, ancho, Fresno, Serrano, Anaheim, Pasilla, Santa Fe, Scotch bonnet, Habanero, preferably the produced fruits are of the bell type or Lamuyo type.
- the cultivated Capsicum plant is a sweet pepper plant.
- a “sweet pepper” in accordance with an embodiment is a pepper plant of which the fruits have an average score of between 0 SHU and 500 SHU, preferably between 0 SHU and 200 SHU, more preferable between 0 SHU and 50 SHU on the Scoville scale, or even 0 SHU.
- the pungency of a sweet pepper is around 0 SHU.
- the amount of capsaicin of pepper fruits is between 0 and around 30 ASTA pungency units, in another embodiment between 0 and 13 ASTA pungency units, or even between 0 and 2 ASTA pungency units.
- the fruits of the pepper plant according to the invention have a SHU between 0 and 100, or between 0 and 500, or between 0 and 500, or between 500 and 1000, or between 1000 and 2000, or between 2500 and 5000 or more.
- the fruits of a pepper plant in accordance with the present invention score between around 5.000 SHU and around 20.000 SHU, or more than around 25.000 SHU, such as between 30.000 and 50.000, or between 50.000 and 100.000, or between 100.000 and 200.000, or between 100.000 and 350.000 SHU, or above 350.000 SHU.
- a cultivated Capsicum plant is a pepper plant of the bell type.
- a bell pepper is a plant of which the mature fruits have a length/width ratio of 1.5 to 0.7 and an average fruit length of at least 7.5 cm, e.g. at least 8 cm, 9, cm, 10 cm or more, e.g. between 7.5 cm and 17.5 cm.
- the mature fruits have a length/width ratio of 1.3 to 0.9 and an average fruit length of at least 7.5 cm, e.g. of 8 cm, 9, cm, 10 cm or more, e.g. a length of between 7.5 and 15 cm.
- the fruit colour can be, e.g., green, red, yellow, orange, white, rainbow (between stages of ripening) and purple.
- North American bell peppers refer to fruits that are about 10 cm long and about 10 cm wide (about 10 cm being a length between 9.5 and 10.5cm), while in Europe bell peppers may be less blocky and more elongated in shape.
- a bell type pepper produces no capsaicin or produces only capsaicin up to 500 SHU or 30 ASTA pungency units. In one preferred embodiment, a bell pepper does not produce capsaicin.
- the length/width ratio of mature pepper fruit of a plant is at most 4, at most 3.0, at most 2.5, at most 2.0, or at most 1.5.
- the width of a mature pepper fruit of a plant is at least 1.5 cm, at least 2.0 cm, at least 3.0 cm, at least 4.0 cm, at least 6 cm, at least 7cm, at least 8 cm, at least 9 cm, at least 10 cm, or more, at its broadest width.
- the width of a mature pepper fruit of a plant is at least 4.0 cm, such as 6.0 cm or more, 7cm or more, or 8 cm, or more cm, at its broadest width and the length/width ratio is at most 3, preferably at most 2.5, preferably at most 2.0, or at most 1.5.
- the cultivated Capsicum plant preferably it has good agronomic and good fruit quality characteristics.
- the cultivated Capsicum plant is in one aspect uniform, both genetically and phenotypically. Especially fruit characteristics are uniform, e.g. regarding shape, color, length and diameter at marketable maturity, flavour, etc.
- Seeds from which the cultivated Capsicum plants, such as the C. annuum plants, as described herein, can be grown are also an embodiment herein.
- seed characteristics are preferably uniform, e.g. seed size, seed color, etc.
- plants of the line or variety comprising QTL11 (or a variant) in homozygous or heterozygous form produce uniform fruits, meaning that there is little variation between fruits of plants grown under the same environmental conditions and when fruits are at the same developmental stage (e.g. for qualitative characteristics at least 98%, 99% or preferably 100% of all plants or plant parts, fruits or seed are identical for the characteristics; for quantitative characteristics at least 90%, 95%, 98% of all plants or plant parts, fruits or seed are identical for the characteristics).
- the cultivated Capsicum plant may be a single cross FI hybrid or an inbred line, comprising QTL11 (or a variant) in homozygous or heterozygous form.
- it is an FI hybrid produced by crossing an (inbred) parent plant comprising QTL 11 (or a variant) in homozygous form with an (inbred) parent plant lacking QTL 11 (i.e. lacking introgression fragment comprising the QTL).
- the FI hybrid is heterozygous for QTL11.
- FI hybrid produced by crossing an (inbred) parent plant comprising QTL11 (or a variant thereof) in homozygous form with an (inbred) parent plant that also comprises QTL11 (or a variant thereof) in homozygous form.
- the FI hybrid is homozygous for QTL11 (or a variant).
- the F 1 hybrid is a sweet pepper type or a hot pepper type, preferably it is a cultivated C. annuum.
- the cultivated Capsicum plant comprising QTL11 produces seedless fruits without pollination, i.e. is parthenocarpic. Such seedless fruits are also encompassed herein.
- the cultivated Capsicum plant comprising QTL 11 is uniform and genetically stable regarding the morphological characteristics of the fruits produced by said plant, e.g. regarding fruit shape, fruit color, etc.
- seeds from which a cultivated Capsicum plant according to the invention can be grown is provided herein, as are fruits harvested from a plant according to the invention. These comprise the QTL in their genome and can therefore be distinguished from other fruits by the presence of one or more of the SNP markers provided herein.
- a further embodiment of the invention is a plant cell, tissue or plant part of a plant or of a seed according to the invention comprising at least one recombinant chromosome 11, wherein said recombinant chromosome 11 comprises an introgression fragment from a wild Capsicum plant and wherein said introgression fragment comprises QTL11 (or a variant) conferring increased A. gossypii resistance compared to a plant lacking the QTL 11 (or a variant).
- a recombinant chromosome 11 comprising an introgression fragment from a wild Capsicum ssp. (said introgression fragment comprising an allele conferring enhanced resistance against Aphis gossypii) for breeding cultivated Capsicum varieties of a domesticated species (especially C. annuum ) having enhanced resistance against Aphis gossypii is encompassed herein.
- said recombinant chromosomes 11 is the recombinant chromosome 11 as found in seeds deposited under accession number NCIMB 43605 or is derived from said recombinant chromosome (e.g. is a smaller fragment of the introgression fragment found in said seeds).
- a chromosome 11 as found in seeds deposited under accession number NCIMB 43605, or in progeny thereof, for generating a cultivated Capsicum plant comprising an introgression fragment on said chromosome 11 is encompassed herein, wherein said introgression fragment confers enhanced Aphis gossypii resistance compared to the control plant lacking said introgression fragment, such as the genetic control or a control breeding line or variety.
- plants grown from seeds deposited under NCIMB43605 (comprising QTL11 in homozygous form) may be used as resistant control.
- susceptible control any variety may be used, such as Mustang FI (Nunhems variety).
- a method for identifying (or detecting) a cultivated Capsicum plant or plant part comprising an introgression fragment on chromosome 11 comprising: a) providing a cultivated Capsicum plant or plant parts or DNA of such plant or plant part, b) screening said plant, plant part or DNA using a molecular marker assay which detects at least one
- SNP marker selected from the group consisting of:
- SNP_01 to SNP_18 preferably SNP_01 to SNP_09, preferably at least SNP_03 and optionally SNP_02 and/or SNP_04, for detecting the introgression fragment on chromosome 11; and optionally c) identifying and/or selecting a plant comprising the resistant donor SNP marker for: i) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the SNP markers of SNP_01 to SNP_18 for detecting the introgression fragment on chromosome 11; or ii) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive markers selected from SNP_01 to SNP_ 18 for detecting the introgression fragment on chromosome 11 ; or iii) at least 1, 2, 3, 4, 5, 6, 7, 8 or more markers of a group, said group consisting of SNP_01 to SNP_06; SNP_06 to SNP_12; SNP_12 to SNP_18; SNP_03 to SNP_10; SNP_10 to SNP_16;
- a method of producing Capsicum F 1 hybrid plants, especially C. annuum F 1 hybrid plants, comprising an introgression fragment conferring enhanced gossypii resistance comprising: a) providing a first inbred Capsicum plant (especially C. annuum) comprising a recombinant chromosome 11 in homozygous or heterozygous form having an introgression fragment comprising QTL11 conferring enhanced gossypii resistance, optionally wherein said introgression fragment is as in NCIMB 43605 or a smaller (sub-) fragment as described, b) providing a second inbred Capsicum plant (especially C. annuum), c) crossing said Capsicum plant of a) w ith said Capsicum plant ofb). d) collecting FI hybrid seeds from said cross.
- a first inbred Capsicum plant especially C. annuum
- a recombinant chromosome 11 in homozygous or heterozygous form
- the second inbred Capsicum plant (especially C. annuum) optionally also comprising a recombinant chromosome 11 in homozygous or heterozygous form having an introgression fragment comprising QTL 11 conferring enhanced A gossypii resistance.
- the FI hybrid seeds collected are also an embodiment of the invention.
- the FI hybrid seeds comprise the introgression fragment comprising QTL 11 in homozygous or heterozygous form.
- a method for generating progeny of NCIMB 43605 comprising: a) growing a plant from seeds deposited under accession number NCIMB 43605; b) selfing said plant one or more times and/or crossing said plant one or more times with another Capsicum (especially C. annuum) plant to generate progeny seeds; c) optionally screening said progeny seeds or plants grown from said seeds or parts of the seeds or plants using a molecular marker assay which detects at least one SNP marker selected from the group consisting of:
- SNP_01 to SNP_18 preferably SNP_01 to SNP_09, preferably at least SNP_03 and optionally SNP_02 and/or SNP_04, for detecting the introgression fragment on chromosome 11; optionally d) identifying and/or selecting a progeny plant comprising the resistant donor SNP marker for: i) at least 1 of the SNP markers of SNP_01 to SNP_18 for detecting the introgression fragment on chromosome 11; or ii) at least 2, 3, or 4 consecutive markers selected from SNP_1 to SNP_ 18 for detecting the introgression fragment on chromosome 11 ; or iii) at least 1, 2, or 3 markers of a group of markers consisting of SNP_01 to SNP_06; SNP_06 to SNP_12; SNP_12 to SNP_18; SNP_03 to SNP_10, SNP_10 to SNP_16 for detecting the introgression fragment on chromosome 11 ; or iv) at least 2, 3
- the plant in step b is preferably a cultivated Capsicum plant, especially C. annuum, such as a sweet pepper, bell pepper or paprika type.
- the method optionally further comprises the step of identifying a progeny plant having enhanced resistance against A. gossypii compared to the control.
- a progeny plant generated by the above method is also an aspect of the invention.
- the progeny plant may comprise a shorter introgression fragment than the one found in NCIMB 43605, which retains the QTL11.
- containers and packages containing or comprising seeds from which plants of the invention can be grown are provided herein. These may be labelled as containing cultivated Capsicum seeds producing enhanced resistance against A. gossypii.
- progeny seeds and progeny plants of plants of the invention are provided, which retain the introgression on chromosome 11 comprising the QTL11, or which comprise a smaller introgression (e.g. derivable from the introgression fragment as is present in NCIMB 43605) which still confers enhanced A. gossypii resistance.
- Progeny may be any generation obtained by selfing a Capsicum plant according to the invention and/or crossing a Capsicum plant according to the invention with another Capsicum plant one or more times.
- Progeny are, therefore, either the generation (seeds) produced from the first cross (FI) or selfing (SI), or any further generation produced by crossing and/or selfing (F2, F3, etc.) and/or backcrossing (BC1, BC2, etc.) one or more selected plants of the FI and/or SI and/or BC1 generation (or plants of any further generation, e.g. the F2) with another Capsicum plant, especially with another cultivated Capsicum plant of a domesticated species.
- Progeny are preferably selected to retain the recombinant chromosome 11 comprising the introgression fragment from a wild Capsicum donor (comprising the QTL11 or a variant thereof). Thus, progeny also have the enhanced A.
- gossypii resistance phenotype preferably at least the same reduced susceptibility to A. gossypii as the plant used in the initial cross or selfing.
- the presence of (or retention of) the introgression fragment comprising the QTL can be determined phenotypically and/or using the molecular marker assay(s) described herein.
- parts of the cultivated Capsicum plants according to the invention are provided.
- Parts include for example cells and cell-cultures, tissue cultures, vegetative plant tissues (leaves, roots, etc.), flowers, pollen, embryos, fruits, parts of fruits, etc.
- the plant parts comprise the introgression fragment on chromosome 11, as described, and as can be detected using one or more of the markers described.
- the regenerated plants comprise the recombinant chromosome 11 and the A. gossypii resistance phenotype.
- a plant cell, tissue or plant part of a plant or of a seed according the invention comprising at least one recombinant chromosome 11, wherein said recombinant chromosome 11 comprises an introgression fragment from a wild Capsicum plant and wherein said introgression fragment comprises an allele or a QTL conferring enhanced A. gossypii resistance.
- cells or tissues comprising a recombinant chromosome 11 described.
- the cells or tissues can be regenerated into a whole Capsicum plant, i.e. the cells are regenerable cells and the tissues comprise regenerable cells.
- vegetative propagations of the plants according to the invention are an embodiment herein.
- a vegetatively propagated cultivated Capsicum plant is provided which comprises a recombinant chromosome 11 as described herein.
- non-propagating cells comprising QTL 11 are encompassed herein, as are tissues comprising such cells.
- a Capsicum fruit harvested from a plant according to the invention is provided.
- cultivated Capsicum plants produce fruits of diverse size, shape and color, any of such fruits comprising QTL 11 are encompassed herein, such as sweet peppers, hot peppers, bell shape, conical, lamuyo, pointed, round, oval, red, green, yellow, etc.
- the cultivated Capsicum plant is a sweet pepper, especially a bell type or lamuyo type and fiuits harvested and optionally processed (e.g. sliced or diced) are provided.
- the cultivated Capsicum plant is a hot pepper and fruits harvested and optionally processed (e.g. sliced or diced) are provided.
- the invention also provides for a food or feed product comprising or consisting of a plant part described herein preferably a Capsicum fruit or part thereof and/or an extract from a plant part described herein.
- the food or feed product may be fresh or processed, e.g., pickled, canned, steamed, boiled, fried, blanched and/or frozen, etc.
- containers such as cans, boxes, crates, bags, cartons, Modified Atmosphere Packaging, films (e.g. biodegradable films), etc. comprising plant parts such as fiuits or fruit parts (fresh and/or processed) described herein are also provided herein.
- plants, plant parts and cells according to the present invention are obtained by a technical method such as a marker assisted selection method as described herein.
- plants, plant parts and cells according to the present invention are not exclusively obtained by means of an essentially biological process as defined by Rule 28(2) EPC.
- a process for the production of plants or animals is essentially biological if it consists entirely of natural phenomena such as crossing or selection as defined by Rule 26(5) EPC.
- the invention provides for a method of producing a new cultivated Capsicum plant which comprises an introgression fragment on chromosome 11 (which confers enhanced A. gossypii resistance) in homozygous or heterozygous form, as described.
- the method comprises crossing a plant of the invention, or a progeny plant thereof, either as male or as female parent, with a second cultivated Capsicum plant one or more times, and/or selfing a Capsicum plant according to the invention, or a progeny plant thereof, one or more times, and selecting progeny from said crossing and/or selfing.
- a method for transferring the recombinant chromosome 11 , comprising the QTL 11 from one (cultivated) Capsicum plant into another (cultivated) Capsicum plant is provided, especially into cultivated Capsicum varieties or breeding lines for which the A. gossypii resistance should be increased.
- the method comprises the steps of: a) providing a first cultivate Capsicum plant, especially cultivated C. annuum, comprising a recombinant chromosome 11 having an introgression fragment comprising a QTL conferring enhanced A. gossypii resistance, preferably in homozygous form, b) providing a second cultivated Capsicum plant, especially a plant having a wild type (non recombinant) chromosome 11, e.g.
- a plant susceptible tori gossypii c) crossing said Capsicum plant of a) with said Capsicum plant of b), d) collecting F 1 hybrid seeds from said cross, and e) optionally selfing the plant grown from said F 1 hybrid seeds to produce F2 seeds or further selfing generations, and optionally selecting the F2 seeds or further selfing generation seeds having the recombinant chromosome 11, and optionally f) optionally breeding further with plants grown from said FI or F2 or fiirther generation selfing seeds to produce a plant having good agronomic characteristics and comprising the introgression fragment in homozygous or heterozygous form.
- the presence or absence of the recombinant chromosome 11, and of the introgression fragment may be determined by one or more of the molecular marker assays described herein and/or by determining whether the ri. gossypii resistance phenotype is significantly increased compared to the plant of step b).
- Further breeding in step f) may comprise selfing, crossing, double haploid production, backcrossing, and combinations thereof (e.g. backcrossing and selfing), etc. Plants, plant parts and seeds obtainable by the above method are encompassed herein.
- the plant of step a) may be a plant grown from seeds deposited under NCIMB 43605, or progeny thereof, or a plant comprising the introgression fragment on chromosome 11 as present in seeds deposited under NCIMB 43605, or a shorter fragment of that fragment / derived from that fragment.
- the inbred Capsicum plant of a) and b) may be homozygous and/or heterozygous for the introgression fragment on chromosome 11, and they may contain introgression fragments of different sizes and/or of different origin, i.e. from different wild Capsicum donors. So, for example the introgression fragment in a) may be the same or a different introgression fragment than in b).
- the inbred Capsicum plant of a) comprises QTL 11 or a variant thereof in homozygous form and/or the inbred Capsicum plant of b) comprises QTL 11 or a variant in homozygous form.
- the introgression fragment comprising QTL11 is the fragment as found in NCIMB 43605 or a smaller fragment derived therefrom (e.g. one of the sub fragments described).
- plants grown from line NCIMB 43605, or progeny thereof, e.g. obtained by selfing and/or crossing and retaining QTL 11 preferably in homozygous form, are used as a parent line for FI hybrid seed production.
- the F 1 hybrid seeds preferably comprise at least one (but preferably two) recombinant chromosome 11 and the FI plants grown from the seeds do therefore produce enhanced A. gossypii resistance compared to the control, e.g. the genetic control.
- embryo rescue may need to be carried out in the above method, especially to obtain progeny of the first interspecific cross.
- the backcross populations may also be selfed, i.e. BC1S1, BC1S2, BC2S1, BC2S2, or others.
- steps b) to f) the presence of the QTL (or the introgression fragment comprising the QTL) may be tested (and plants may be selected) by carrying out a molecular marker assay as described elsewhere herein.
- the method for producing a cultivated Capsicum plant comprising an introgression fragment on chromosome 11, wherein said introgression fragment comprises QTL 11 or a variant thereof comprises the steps: a) providing a first cultivated Capsicum plant, b) providing a second wild Capsicum comprising one or more of the SNP markers provided herein, i.e.
- accession from b) comprising at least one or more of the following markers: a) the GG (homozygous) or GX (heterozygous) genotype for SNP_01 at nucleotide 101 of SEQ ID NO:
- step B) C) and D) also other molecular marker tests described elsewhere herein can be used.
- this method one can, thus, screen wild Capsicum accessions for the presence of one or more of the markers and, thus, the presence of QTL 11 (or a variant thereof) and introgress the QTL into cultivated Capsicum plants. Plants and seeds obtained by this method are also an embodiment of the invention.
- a method for identifying a cultivated Capsicum plant comprising an introgression fragment on chromosome 11, wherein said introgression fragment comprises a A. gossypii resistance QTL, is provided, said method comprising: screening a cultivated Capsicum plant or a population of cultivated Capsicum plants or parts of such Capsicum plants (e.g.
- any of the molecular marker tests described elsewhere herein can be used.
- a method for detecting whether a cultivated Capsicum plant comprises an introgression fragment on chromosome 11 , wherein said introgression fragment comprises QTL 11 comprising: a) providing cultivated Capsicum plant or a plant part, b) screening said plant or said plant part (or DNA obtained from said plant or plant part) using a molecular marker assay which detects at least one (preferably at least 2, 3, 4, 5 or more) SNP marker selected from the group consisting of: SNP_01 to SNP_18, preferably at least 1, 2, 3, 4, 5, 6, 7, 8 or more SNP markers of SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02 to SNP_08, SNP_01 to SNP_06, SNP_02 to SNP_06, SNP_01 to SNP_05, SNP_02 to SNP_05, SNP_01 to SNP_04, SNP_02 to SNP_04
- the screening of step b) preferably includes at least the marker screening for SNP_03 and optionally for SNP_02 and/or SNP_04.
- a method for identifying a Capsicum plant comprising QTL11 comprising: a) screening a Capsicum plant using a molecular marker assay which detects the SNP marker haplotype or genotype of the plant for one or more markers of SNP_01 to SNP_09; b) optionally identifying and/or selecting a plant comprising the resistant donor SNP marker haplotype or genotype for: i) at least 1, 2, 3, 4, 5, 6, or 7 of the SNP markers of SNP_01 to SNP_09; preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04, or ii) at least 2, 3, 4, 5, 6, or 7 consecutive markers selected from SNP_01 to SNP_09, preferably at least for SNP_03 and optionally for SNP_02 and/or SNP_04, or iii) at least for SNP_03 and optionally for SNP_02 and/or SNP_04, wherein the donor SNP marker haplotype is a Gu
- markers herein, therefore also the marker genotype and/or the marker haplotype is referred to. So detecting one or more SNP markers, refers to determining the plant (or plant part) genotype or plant haplotype for the marker(s).
- Also encompassed herein is a method for producing a cultivated Capsicum plant comprising an introgression fragment on chromosome 11, wherein said introgression fragment comprises QTL11, comprising: a) providing a first cultivated Capsicum plant lacking an introgression fragment comprising QTL11, b) providing a second cultivated Capsicum plant selected from plants grown from seeds deposited under accession number NCIMB 43605 or progeny thereof, c) crossing said plant of a) with said plant of b), d) collecting FI seeds from said cross and optionally selfing said FI plants one or more times to produce an F2 or F3 or further selfing population, e) optionally backcrossing the F 1 plant or an F2 or F3 or further selfing plant to the plant of a) to produce a backcross population, f) optionally selfing the backcross population one or more times, g) identifying a F 1, F2, F3, further selfing or backcross plant which comprises one or more or all of the SNP marker haplotype
- a method of producing FI hybrid plants comprising: a) providing a first inbred Capsicum plant comprising at least one recombinant chromosome 11 having an introgression fragment comprising QTL11, wherein said introgression fragment is the fragment as found in NCIMB 43605, or a shorter fragment of that introgression fragment retaining QTL 11, b) providing a second inbred Capsicum plant with or without a recombinant chromosome 11, c) crossing said plant of a) with said plant of b), d) collecting FI hybrid seeds from said cross.
- a method for generating progeny of NCIMB43605 retaining QTL 11 comprising: a) growing a plant from seeds deposited under accession number NCIMB43605; b) selfing said plant one or more times or crossing said plant one or more times with another cultivated Capsicum plant to generate progeny seeds; c) optionally screening said progeny seeds or plants grown from said seeds or parts of the seeds or plants using a molecular marker assay which detects at least one SNP marker disclosed herein; d) optionally identifying and/or selecting a progeny plant comprising at least one, two, three or more of the SNP markers indicative of the introgression fragment comprising the QTL 11 (as described elsewhere herein), especially one or more of SNP_01 to SNP_18, preferably at least 1, 2, 3, 4, 5, 6, 7, 8 or more SNP markers of SNP_01 to SNP_09, SNP_02 to SNP_09, SNP_01 to SNP_08, SNP_02
- QTL11 is present in the progeny in d), preferably in homozygous form.
- the A. gossypii resistance in e) is preferably at least the same resistance level as for plants grown from NCIMB43605 when grown under the same conditions.
- a method for generating progeny of NCIMB 43605 comprising: a) growing a plant from seeds deposited under accession number NCIMB 43605; b) selfing said plant one or more times or crossing said plant one or more times with another Capsicum plant to generate progeny seeds; c) optionally screening said progeny seeds or plants grown from said seeds or parts of the seeds or plants using a molecular marker assay which detects at least one SNP marker selected from the group consisting of:
- SNP_01 to SNP_18 preferably SNP_01 to SNP_09 for detecting the introgression fragment on chromosome 11; d) optionally identifying and/or selecting a progeny plant comprising the donor SNP for: i) at least 1, 2, 3, 4, 5, 6, 7, 8 or more of the SNP markers of SNP_01 to SNP_18, or preferably of SNP_01 to SNP_09, for detecting the introgression fragment on chromosome 11, said progeny plant preferably comprises at least the donor SNP nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_04; or ii) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive markers selected from SNP_01 to SNP_18, or preferably of SNP_01 to SNP_09, for detecting the introgression fragment on chromosome 11, said progeny plant preferably comprises at least the donor SNP nucleotide for SNP_03 and optionally for SNP_02 and/or SNP_
- a progeny plant generated by any of the above methods is also an aspect of the invention.
- the invention encompasses the use of a recombinant chromosome 11 comprising an introgression fragment from a wild Capsicum donor, said introgression fragment comprising a A. gossypii resistance QTL, for breeding Capsicum varieties having enhanced resistance against A. gossypii.
- chromosome 11 as found in seeds deposited under accession number NCIMB43605 or progeny thereof for generating cultivated Capsicum plant comprising an introgression fragment of said chromosome 11.
- a further aspect herein is the use of any of the SNP markers, SNP haplotypes or genotypes, or sequences provided herein for screening plants or plant parts (or DNA thereof), for marker assisted selection of plants or plant parts, for the identification and/or selection of wild or cultivated plants and for breeding plants comprising QTL 11 or a variant thereof.
- the SNP markers SNP_01 to SNP_18, and also the SEQ ID NOs 1 to 18 and sequences comprising at least 95% sequence identity to any of these are one aspect herein, for use in e.g. analyzing wild or cultivated Capsicum plants or plant parts or for use in markers assays (e.g. SNP genotyping assays).
- a modified (recombinant) cultivated Capsicum chromosome 11 is provided herein, which comprises an introgression fragment of a wild Capsicum, as described throughout the specification.
- the recombinant chromosome is isolated from its natural environment. In another aspect it is in a plant cell, especially in a Capsicum cell, especially in a cultivated Capsicum cell. Also an isolated part of the recombinant chromosome comprising the QTL is provided herein.
- a recombinant nucleic acid molecule which comprises an A. gossypii resistance QTL (or the one or more causal genes underlying the QTL) according to the invention.
- the resistance QTL is detectable by one or more of the molecular marker assays described herein.
- a DNA vector is provided comprising the recombinant DNA.
- the recombinant DNA molecule or DNA vector may be an isolated nucleic acid molecule.
- the DNA comprising the resistance QTL or the one or more causal genes underlying the QTL may be present in a microorganism, such as a bacterium (e.g. Agrobacterium).
- Such a (isolated or extracted) nucleic acid molecule and/or of such a recombinant chromosome or part thereof for generating plant cells and plants comprising an A. gossypii resistance QTL or gene (or allele) is encompassed herein.
- it may be used to generate transgenic plant cells and transgenic plants, e.g. Capsicum cells, Capsicum plants and parts (e.g. fruits) comprising the resistance QTL or causal gene or allele and the plant comprises an enhanced gossypii resistance phenotype.
- transgenic plant cells e.g. transgenic Capsicum cells, comprising in their genome a recombinant chromosome 11 as described and/or a recombinant nucleic acid molecule comprising an A. gossypii- resistance conferring gene or allele are also an embodiment of the invention.
- the DNA molecule comprising the gene or allele is stably integrated into the Capsicum genome.
- the resistance conferring gene or allele may also be cloned and a chimeric gene may be made, e.g. operably linking a plant expressible promoter to the allele.
- a chimeric gene may be introduced into a plant cell and the plant cell may be regenerated into a whole plant to produce a transgenic plant.
- the transgenic plant is a Capsicum plant.
- transgenic plants especially transgenic cultivated Capsicum plants, comprising a resistance conferring gene or allele and having increased resistance against A. gossypii are provided herein.
- cells or cell cultures comprising a recombinant chromosome 11 according to the invention are an embodiment, independent whether the recombinant chromosome 11 is introduced by transgenic methods or by breeding methods.
- the cells are e.g. in vitro and are regenerable into plants comprising the recombinant chromosome 11 of the invention.
- a representative sample of seeds of a C. annuum line of the sweet bell pepper type, designated APHIGO-R, comprising an introgression fragment comprising QTL11 in homozygous form was deposited by Nunhems B.V. on 7 May 2020 at the NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksbum Aberdeen, Scotland AB21 9YA, UK) according to the Budapest Treaty, under the Expert Solution (EPC 2000, Rule 32(1)). Seeds were given the following deposit numbers NCIMB 43605 (APHIGO-R).
- the Applicant requests that samples of the biological material and any material derived therefrom be only released to a designated Expert in accordance with Rule 32(1) EPC or related legislation of countries or treaties having similar rules and regulation, until the mention of the grant of the patent, or for 20 years from the date of filing if the application is refused, withdrawn or deemed to be withdrawn.
- At least 20 plants per genotype was analyzed in any phenotyping test, together with appropriate controls (resistant donor, susceptible controls). For insect rearing the susceptible variety Mustang FI or a susceptible C. baccatum was used.
- Aphis sossvpii insect rearing Pepper plants (2-3 true leaves) were placed in a rearing cage (Bugdorm). A leaf disk of heavily infected plant material was placed in the new cage. Approximately 12 - 15 days after infection (depending on the infection intensity respectively on the condition of the forage crops) a new rearing group was prepared and consumed material was removed.
- Aphis sossvpii inoculation and scoring Per plant, two clip-on cages were placed. In each cage, 4 to 5 adults were placed and left for 24 hours. After this period all adults were removed and only 5 one-day old nymphs were left (all excess as also removed). The nymphs were left in the cages for a period of 8 days after which the number of adults and/or the number nymphs were counted.
- the clip-on cage experiment thus measures the proportion of aphids that reach adulthood (survival) and the reproduction (new nymphs) of aphids.
- Aphis sossypii insect rearing Pepper plants (2-3 true leaves) were placed in a rearing cage (Bugdorm). A leaf disk of heavily infected plant material was placed in the new cage. Approximately 12 - 15 days after infection (depending on the infection intensity respectively on the condition of the forage crops) a new rearing group was prepared and consumed material was removed.
- Aphis sossvpii inoculation and scoring 6 to 8 weeks old pepper plants were placed in a complete randomized design in a greenhouse. All the plants were covered together with an insect proof cloth and each plant was infested with a leaf disk harboring at least 50 insects. The population was left to grow and multiply and 10 dpi (days post inoculation) the plants were assessed using a visual scale. Each plant was assessed on a 1 to 9 scale:
- the population growth test measures the aphid ability to establish, colonize and reproduce on plants.
- QTL mapping Interval mapping was performed. First a Genome Wide Mapping (GW) was performed using a marker density of approximately 1 marker every 15 cM. Once QTLs were identified a second round of mapping or saturation mapping was performed by adding 24 markers within the QTL intervals identified by GW mapping.
- QTL Validation For the QTL validation BCISn families were used. Individual plants were genotyped using flanking and peak markers identified during the saturation mapping. Phenotype on individual plants was obtained as indicated above. An ANOVA test followed by a Tukey HSD test was performed to test for the different allelic states within a family. ANOVA and Tukey HSD test were performed.
- Example 2 Results A QTL conferring improved level of tolerance (or resistance, reduced susceptibility) to aphid ( Aphids gossypii ) was mapped, which reduces the average number of nymphs and/or adults.
- the QTL was found on chromosome 11 (QTL11) and was derived from the wild donor Capsicum baccatum. Results of clip-on cage experiments are shown in Figure 1 and Table A. Average number of nymphs was significantly lower on the C. annuum plants comprising QTL11 compared to the susceptible control lacking the QTL. The same effect was seen for the average number of adults.
- Results of the population growth test showed that aphids showed reduced reproduction (population growth) on the QTL 11 containing plants compared to the control plants lacking the QTL and the susceptible parent lacking the QTL.
- the QTL11 from the wild donor accession was mapped to the region shown in Figure 2, with the markers in Table 1 below linked to the QTL.
- sequence flanking the SNP nucleotide to the right and to the left is the reference genome sequence and not the sequence of the QTL donor.
- this encompasses in one aspect that there are e.g. one, two or three nucleotide differences in the flanking sequences of the wild donor, i.e. it encompasses the donor SNP nucleotide (e.g. G at nucleotide 101 of SEQ ID NO: 1) being present in a sequence comprising at least 95% sequence identity to SEQ ID NO: 1, i.e. G at nucleotide 101 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 1.
- Table 2
- Genomic DNA is double stranded, comprising a forward strand (plus strand) and a reverse strand (minus strand), which are complementary to one another.
- the DNA sequences for some of the SNP markers may be the reverse strand sequences (- strand) with respect of the reference genome sequence of pepper. This can be seen by e.g. Blast analysis of the sequence against the reference genome database, resulting in an alignment with either the forward strand, or plus strand (wherein nucleotide numbering is counting upwards) or with the reverse strand / complement strand whereby nucleotide numbering is counting downwards. It is understood that this is just a matter of naming.
- nucleotide T for SNP_02 at nucleotide 101 in SEQ ID NO: 2 corresponds to the nucleotide A for SNP_02 at nucleotide 101 of the complementary sequence of SEQ ID NO: 2.
- the donor SNP nucleotide in a reverse strand sequence thus also comprises a reference to the complement donor SNP nucleotide in the complement/forward strand of the sequence (and vice versa, reference to a donor nucleotide in a forward strand sequence can also be referred to by referring to the complement donor nucleotide in the reverse strand).
- the assay one can design the assay to detect e.g. the T in SEQ ID NO: 2 or the A in the complementary strand of SEQ ID NO: 2. This applies to all SNP marker sequences.
- Fine mapping was carried out in order to determine in which region QTL 11 was found.
- a selfed backcross population of recombinant plants was grown and phenotyped as described above and a large set of SNP markers was used to genotype the plants.
- QTL11 lies in between SNP_01 at nucleotide 101 of SEQ ID NO: 1, or SNP_02 at nucleotide 101 of SEQ ID NO: 2, and SNP_06 at nucleotide 101 of SEQ ID NO: 6 and comprises the donor SNP nucleotide for one or more of SNP_02, SNP_03, SNP_04 and/or SNP_05.
- QTL11 lies in between SNP_02 and SNP_06 and comprises at least the donor SNP nucleotide for SNP_03, optionally also for SNP_02 and/or SNP_04.
- QTL11 lies in between SNP_02 and SNP_04 and comprises at least the donor SNP nucleotide for SNP_03, optionally also for SNP_02 and/or SNP_04.
- SNP 03 had the highest LOD score for both phenotypes mapped, which were ‘new nymphs’ as described above (clip-on cage experiment, no choice test) and population growth (free choice experiment) as described above. SNP_03 is thus the peak marker, most closely linked to the phenotype.
- N/A not included, * peak marker QTL11, therefore, lies in between SNP_01 and SNP_09, especially in between SNP_02 and SNP_06, or between SNP_02 and SNP_04, with SNP_03 being most closely linked to QTL11.
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