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RU-2861559-C2 - RESISTANCE TO DIDYMELLA BRYONIAE INTERNAL FRUIT ROT IN CUCUMIS SATIVUS PLANTS

RU2861559C2RU 2861559 C2RU2861559 C2RU 2861559C2RU-2861559-C2

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to a Cucumis sativus plant of the sativus variety, wherein the plant's genome is modified to increase the average gene expression of eight OGD genes, OGD1 - OGD8, encoding 2-oxoglutarate Fe(II)-dependent oxygenase (OGD) proteins, by at least 1.3 times compared to the average gene expression of the eight OGD genes in a susceptible wild-type plant containing two copies of each OGD gene, as well as to its seed and fruit. EFFECT: increasing resistance to symptoms of internal fruit rot caused by Didymella bryoniae in cultivated cucumbers. 9 cl, 8 dwg, 8 tbl, 7 ex

Inventors

  • LIBERTI, Daniele
  • KOELEWIJN, Hans, Peter
  • BEENDERS, Frank
  • REINIERS, Laura
  • VRIEZEN, Wim
  • MERTENS, Lieke

Dates

Publication Date
20260506
Application Date
20210315
Priority Date
20200318

Claims (20)

  1. 1. A Cucumis sativus cultivar sativus plant having enhanced resistance to internal fruit rot symptoms caused by Didymella bryoniae , wherein the genome of the plant has been modified to increase the average gene expression of eight OGD genes, OGD1 through OGD8, encoding 2-oxoglutarate Fe(II)-dependent oxygenase (OGD) proteins by at least 1.3-fold compared to the average gene expression of the eight OGD genes in a susceptible wild-type plant containing two copies of each OGD gene, wherein the eight OGD genes are genes encoding
  2. - an OGD1 protein comprising an amino acid sequence according to SEQ ID NO: 52 or a sequence having at least 95% sequence identity with SEQ ID NO: 52,
  3. - an OGD2 protein comprising the amino acid sequence according to SEQ ID NO: 53 or a sequence having at least 95% sequence identity with SEQ ID NO: 53,
  4. - an OGD3 protein comprising the amino acid sequence according to SEQ ID NO: 54 or a sequence having at least 95% sequence identity with SEQ ID NO: 54,
  5. - an OGD4 protein comprising an amino acid sequence according to SEQ ID NO: 55 or a sequence having at least 95% sequence identity with SEQ ID NO: 55,
  6. - an OGD5 protein comprising an amino acid sequence according to SEQ ID NO: 56 or a sequence having at least 95% sequence identity with SEQ ID NO: 56,
  7. - an OGD6 protein comprising the amino acid sequence according to SEQ ID NO: 57 or a sequence having at least 95% sequence identity with SEQ ID NO: 57,
  8. - an OGD7 protein comprising an amino acid sequence according to SEQ ID NO: 58 or a sequence having at least 95% sequence identity with SEQ ID NO: 58,
  9. - an OGD8 protein comprising an amino acid sequence according to SEQ ID NO: 59 or a sequence having at least 95% sequence identity with SEQ ID NO: 59,
  10. and/or wherein the plant genome is modified to increase the average gene expression of a gene encoding an HR-like protein by at least 1.3 times compared to the average gene expression of said gene in a susceptible wild-type plant containing two copies of the gene, wherein said gene is a gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 60 or a sequence having at least 95% sequence identity with SEQ ID NO: 60,
  11. wherein said plant comprises in its genome a duplication of a region of chromosome 3 flanked by and containing SEQ ID NO: 83 and SEQ ID NO: 84, wherein said region contains all of said OGD genes and said gene encoding said HR-like protein, and
  12. wherein said resistance to the symptoms of internal fruit rot caused by Didymella bryoniae is increased compared to a plant that does not have said duplication.
  13. 2. The plant of claim 1, wherein the duplication is a duplication introgressed from a wild-type cucumber, or induced by targeted modification of the genome, or introduced by transformation.
  14. 3. The plant of claim 1, further comprising an introgression fragment from a wild-type cucumber on chromosome 5 containing the quantitative trait locus QTL5.1, wherein the introgression fragment containing QTL5.1 contains a haplotype of at least 3 markers selected from:
  15. - guanine for SNP_12 at nucleotide 51 of the sequence according to SEQ ID NO: 12 or at nucleotide 51 of the sequence having at least 97% identity with SEQ ID NO: 12,
  16. - adenine for SNP_45 at nucleotide 51 of the sequence according to SEQ ID NO: 45 or at nucleotide 51 of the sequence having at least 97% identity with SEQ ID NO: 45,
  17. - adenine for SNP_13 at nucleotide 51 of the sequence according to SEQ ID NO: 13 or at nucleotide 51 of the sequence having at least 97% identity with SEQ ID NO: 13,
  18. - guanine for SNP_14 at nucleotide 51 of the sequence according to SEQ ID NO: 14 or at nucleotide 51 of the sequence having at least 97% identity with SEQ ID NO: 14,
  19. - thymine for SNP_46 at nucleotide 51 of the sequence according to SEQ ID NO: 46 or at nucleotide 51 of the sequence having at least 97% identity with SEQ ID NO: 46,
  20. - thymine for SNP_47 at nucleotide 51 of the sequence according to SEQ ID NO: 47 or at nucleotide 51 of the sequence having at least 97% identity with SEQ ID NO: 47,

Description

The present invention relates to the field of cucumber breeding and cucumber genome modification. In one aspect, there is provided the introgression of a quantitative trait locus (QTL) located on chromosome 5 of a cultivated cucumber genome and/or the introgression of a QTL on chromosome 3 of a cultivated cucumber genome, which can be used to improve resistance to internal fruit rot caused by the ascomycete Didymella bryoniae (abbreviated DB) in cultivated cucumbers (Cucumis sativus cv. sativus), such as long cucumbers, gherkins (e.g., American pickling, European pickling), salad cucumbers (e.g., American slicing), short cucumbers, European greenhouse cucumbers, Beit Alpha type cucumbers, oriental trellis (also referred to as 'burpless') type cucumbers, Asian cucumbers (which can be further subdivided into different types such as Indian variegated cucumber, Chinese long cucumber, Korean cucumber and Japanese cucumber, the first of which belongs to the Indian group of cucumbers, and the last three to the East Asian group of cucumbers). Furthermore, the causal genes underlying QTL3 have been identified, allowing plants containing modifications of these genes to be created, for example, through targeted genome editing, mutagenesis, or transformation. Plants and plant parts containing such modifications constitute one aspect of the present invention. The QTL on chromosome 5 is referred to herein as QTL5.1, and the QTL on chromosome 3 is referred to as QTL3.1. In one aspect, both are introgression from the same wild cucumber, called MYCR3 (proprietary name). This plant was used for mapping and introgression of QTL into the European long cucumber type, into different genetic backgrounds (using different repeat parents). A representative sample of seeds containing both QTLs was deposited, and from the deposit or from the progeny of this deposit, one or both QTLs can be easily transferred to any other cucumber type, such as short cucumber types, or to any other line or variety of another long cucumber type. Alternatively, other donors may be identified that contain the same QTLs, such as those containing the same SNP haplotypes for QTL3.1 and/or for QTL5.1 or the same SNP haplotype for at least several, such as at least 10 or more SNP markers, especially for at least 10 or more consecutive SNP markers, or that contain a duplication containing QTL3.1, such as a duplication of SEQ ID NO: 72 (or a genomic region containing at least 95% sequence identity with SEQ ID NO: 72), or a duplication of the genomic region beginning with (and including) SEQ ID NO: 83 and ending with (and including) SEQ ID NO: 84 of chromosome 3. Seeds containing both introgression fragments in homozygous form, i.e. QTL3.1 and QTL5.1 in homozygous form, as well as containing donor SNP markers/sequences indicating introgression fragments in homozygous form, were deposited under NCIMB accession number 43530. Genetic control seeds lacking both introgression fragments were deposited under NCIMB accession number 43531. QTL5.1 was initially mapped to SNP markers SNP_01 to SNP_18 and then fine-mapped to the region associated with SNP_12 to SNP_15, with the addition of additional markers to the region. See Fig. 6. Thus, QTL5.1 is located between SNP_12 and SNP_15 on chromosome 5. QTL3.1 was initially mapped to the region linked to SNP_19-SNP_42 and was fine-mapped to SNP_35 and 659 nucleotides downstream of SNP_36. This means that QTL3.1 is closely linked to SNP_35 and SNP_36 on chromosome 3. The entire region was sequenced, which showed that QTL3.1 (presented herein as SEQ ID NO: 72) was duplicated on chromosome 3. The duplication of QTL3.1 was part of a larger duplication of approximately 150 kb (effectively 147,292 bases) on chromosome 3, see the gray bars in Fig. 4, where the beginning and end of the large duplicated region are presented herein as SEQ ID NO: 83 and SEQ ID NO: 84, respectively. Thus, the resistant plant contained QTL3.1 twice on chromosome 3, as shown in Fig. 4. Since the resistant plant whose seeds were deposited was homozygous for QTL3.1, it effectively contained QTL3.1 four times, two copies on each chromosome 3. In contrast, the susceptible plant contained only one copy of QTL3.1 on chromosome 3 (and in the homozygous deposited seeds, therefore, two copies of QTL3.1). This structural variation for QTL3.1, detected by sequencing, means that the nomenclature used in this document has, in one respect, a revised meaning. When referring to a resistant plant “containing QTL3.1” in homozygous or heterozygous form, compared to a susceptible plant “not containing QTL3.1,” this actually refers, in one respect, to a resistant plant “containing a duplication of QTL3.1” in homozygous or heterozygous form, compared to a susceptible plant “not containing a duplication of QTL3.1.” Sequencing of the QTL3.1 region revealed that there are 9 genes in the precisely mapped region of QTL3.1. Expression analysis further revealed that all these genes were