Patent Grant
PP34398
Latin name of the genus and species of the plant claimed: Musa acuminata.
Variety denomination: ‘QCAV-4’.
The present invention relates to a new and distinct cultivar of banana plant named ‘QCAV-4’. The new plant resulted from transformation of parent Cavendish Grand Nain (unpatented) by T-DNA insertion and selection. A resulting transgenic plant named ‘QCAV-4’ was selected when growing in a cultivated area in Lambells Lagoon, Northern Territory, Australia.
‘QCAV-4’ is a transgenic cultivar produced from Cavendish Grand Nain. For initial transformation, embryogenic cell suspensions (ECS) were generated from immature male flowers from the bell (flower) of Cavendish Grand Nain. The bells were collected in North Queensland, Australia and indexed for virus infection. The ECS were transformed using Agrobacterium mediated transformation. The transformation cassette included a selectable marker gene, neomycin phosphotransferase (NPT II). The resistance gene was a gene isolated from Musa acuminata subsp. malaccensis which is resistant to Fusarium wilt tropical race 4 (TR4). The resistance gene was under the control of the nos promoter. Potentially transformed cells were placed on kanamycin to select NPT II resistant cells. These were then regenerated into whole plantlets and multiplied. Presence of the transgenes were confirmed by PCR. Multiplied plantlets were transferred to a farm in Lambells Lagoon, Northern Territory, Australia and acclimatized in a screenhouse. These plants together with appropriate controls were planted into a plot where Cavendish bananas had been previously grown and had been severely affected by Fusarium wilt TR4. The plot was “seeded” further with pseudostem segments from infected Cavendish plants. Plants were regularly inspected for TR4 symptoms over a three-year period. Multiple independent transformed lines demonstrated strong resistance to TR4 as compared to the parental Cavendish Grand Nain, which is highly susceptible. Morphological characteristics of plants and fruit were assessed, bunch weight was measured, and molecular analysis was performed. One line was selected based on morphological and molecular analysis, and named ‘QCAV-4’.
The ‘QCAV-4’ cultivar is distinguished from other banana varieties, including the parent, by having a strong resistance phenotype to Fusarium wilt tropical race 4 (TR4). It is substantially phenotypically identical to its parent in the absence of disease pressure.
Asexual reproduction of ‘QCAV-4’ by tissue culture in Brisbane City, Queensland, Australia in combination with field assessment in Lambells Lagoon, Northern Territory, Australia, shows that the foregoing characteristic resistance to Fusarium wilt TR4 reproduces true to type.
The following detailed description concerns progeny lines asexually propagated from the original line by tissue culture.
The colors of an illustration of this type may vary with lighting and other conditions under which conditions and, therefore, color characteristics of this new cultivar should be determined with reference to the observations described herein, rather than from these illustrations alone.
The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter code for amino acids, as defined in 37 C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file, created on Dec. 14, 2021, 12 KB, which is incorporated by reference herein. In the accompanying sequence listing:
SEQ ID NOs: 1-7 are new ORF sequences found in ‘QCAV-4’ that resulted from the transgenic event.
The following detailed description of ‘QCAV-4’ is based on observations of plants that are approximately 25 months old. The ‘QCAV-4’ plants have been observed growing in a cultivated area in Lambells Lagoon, Northern Territory, Australia. Certain characteristics of this cultivar, such as growth and color, may change with changing environmental conditions (such as, light, temperature, moisture, nutrient availability, or other factors). Color descriptions and other terminology are used in accordance with their ordinary dictionary descriptions, unless the context clearly indicates otherwise.
Scientific name: Musa acuminata ‘QCAV-4’
Parentage: Cavendish Grand Nain
Plant:
In the absence of significant disease pressure, ‘QCAV-4’ appears to be essentially phenotypically identical to the wild type parent ‘Cavendish Grand Nain’ (unpatented) (Table 1). This includes in relation to immature and mature plant characteristics, fruit characteristics, and yield.
However, a clear phenotype is observable under pressure from Fusarium wilt tropical race 4 (TR4).
In March 2018, an expanded field trial was planted which included 50 replicates of each of the four events from Trial 1, in 10×5 randomized plot design. In addition to recording disease incidence, detailed agronomic information such as bunch weight, number of fingers on the top hand and crop cycling time is also collected. Since the trial began, agronomic data for the plant crop and at least two ratoon crops were collected. The trial is ongoing. Based on the results of these field trials and molecular characterisation, ‘QCAV-4’ was selected.
The disease status of plants is assessed by the presence of characteristic disease symptoms (both external and internal) and by molecular testing of vascular tissue for the presence of the fungal pathogen TR4. The plants are inspected on a weekly basis and plants showing the characteristic external symptoms of the disease identified. About 1-2 weeks later, the pseudostem of these plants is cut and examined for the presence of the highly characteristic internal vascular discolouration associated with TR4 infection. DNA is extracted from the infected vascular tissue, and a highly sensitive PCR test is used to detect the presence of TR4, and this is confirmed by sequencing. The TR4 fungus from discoloured vascular tissue is obtained and DNA extracted and analysed using PCR to confirm the presence of TR4 (and also by sequencing).
As shown in Table 2, ‘QCAV-4’ can remain largely disease-free under the same conditions of TR4 pressure leading to greater than 80% infection rates in wild type ‘Cavendish Grand Nain’.
Height of about 180 to 250 cm—shorter than Giant Cavendish and taller than Dwarf Cavendish cultivars.
Moderate adult pseudostem width.
Relatively large bunch size.
Moderate fruit size.
Solid green leaf colour.
Southern Analysis:
Genomic DNA was extracted from ‘QCAV-4’ and wild type (non-transformed) Cavendish Grand Nain. The DNA was digested with a restriction enzyme, electrophoresed through an agarose gel, transferred to a membrane, and probed with a labelled RGA2 probe.
As shown in
Similar experiments were performed with wild type Cavendish Grand Nain (parent) and independent transgenic lines ‘RGA2-2’, ‘RGA2-3’, ‘RGA2-4’ (clonal progenitor of ‘QCAV-4’), ‘RGA2-5’ and ‘RGA2-7’. As shown in
Genome sequencing:
Long Read Sequencing of Event ‘QCAV-4’
High molecular weight genomic DNA (with average fragment size >50 Kb) was isolated from young in vitro leaf tissue of ‘QCAV-4’ using GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, USA). For long-read sequencing on PacBio Sequel II platform (Novogene, China), a size-selected library with an insert size of 20 Kb was generated. A total of ˜75 Gbp data was obtained in CLR mode (4.9 M reads with a read length N50 of 17,973 bp). This corresponds to ˜42× coverage of the Cavendish genome at the haplotype level. SAN-3 binary vector T-DNA sequence was used to filter out long-reads from the total genomic pool. About 80 long-reads which mapped onto the T-DNA sequence were then assembled using Flye plugin in Geneious Prime 2020. A single ˜27 kb T-DNA insertion locus was assembled. This sequence, along with 5 kb flanking sequence, was polished using ‘RGA2-4’ genomic Illumina short reads (previously generated using Novaseq 6000) to correct a few Flye assembly errors (short indels). Nucleotide BLAST using the two flanking sequences of this T-DNA locus revealed that the insertion of T-DNA locus has occurred in chromosome 6 of the banana genome.
Details of the T-DNA Insertion
Event ‘QCAV-4’ contains a complex T-DNAs insert of 26,849 bp at a single genomic location on chromosome 6 between position 29,939,311 and 29,939,427 (−strand) creating a 116 bp deletion. The insert is located in an intergenic region between two intact predicted genes: Ma06_t28200.1 (a putative Malectin_like domain-containing protein) at position chr06:29,931,700..29,937,001 (+strand) and Ma06_t28210.1 (a malectin_like domain-containing protein) at position chr06:29,944,119..29,947,729 (+strand). Both genes are not affected by the insertion and it is not predicted that the insertion will affect their expression.
The insert itself is composed of three full and functional copies of the 6702 bp T-DNA (T-DNA 1 to 3, see
New Open Reading Frames (ORFs) Analysis
The analysis identified 7 new ORFs (SEQ ID NOS: 1-7), all originating from these rearranged genome/T-DNA and inter T-DNA junctions. New ORFs AA sequences as follows:
Assessment of the Expression of the Seven New ORFs
To assess the expression potential of the seven newly identified ORFs, two RNAseq Illumina libraries were used. Root and leaf RNAseq libraries containing 274,556,348 and 268,119,840 reads, respectively were mapped to the reconstructed insertion locus. From this analysis, 1,029,853 and 781,191 reads originating from the leaf and root RNAseq dataset respectively mapped to the insert sequence. No read from either library mapped continuously across any of the seven newly identified ORFs, confirming the lack of mRNA originating from them in event QCAV-4.
Bioinformatic Assessment of the Allergenicity Potential the Seven New ORFs
In silico analyses performed (see below) to compare amino acid sequence of each new ORF to known allergenic proteins in the Food Allergy Research and Resource Program (FARRP) dataset, which is available through AllergenOnline (University of Nebraska). Full length sequence (E value <10−5), 80-mer sliding window (35% homology with E value <10−4) and 8-mer exact match searches identified no sequences similarity between any of the 7 new ORFs and known allergens in the database.
Bioinformatic Assessment of the Toxicity Potential of the Seven New ORFs
Potential structural similarities shared between the seven new ORFs and sequences in a protein toxin database were evaluated using the Basic Local Alignment Search Tool (BLAST) available within the Geneious program.
A blastp search using the BLOSUM45 similarity scoring matrix and the amino acid sequence from the seven new ORFs as the query sequence did not return any accessions of biological significance from the toxin database with an E-score acceptance criteria lower than 1×10−4.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6133035 | Engler et al. | Oct 2000 | A |
| 7601887 | Dale et al. | Oct 2009 | B2 |
| Entry |
|---|
| “Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4,” Dale et al., Nature Communications,8: 1496, |DOI: 10.1038/s41467-017-01670-6/www.nature.com/naturecommunications, pp. 1-8, published Nov. 14, 2017. |
| Plant Variety Gazette of the Philippines Plant Variety Protection Office, vol. 32, Jun. 28, 2020 (10 pages). See p. 4. |
| Plant Varieties Journal, vol. 33, No. 3, Nov. 20, 2020 (416 pages). See p. 10. |
| Declaration of Timothy Fitzgerald, Ph.D., executed on Dec. 11, 2021. |
| Number | Date | Country | |
|---|---|---|---|
| 20210400858 P1 | Dec 2021 | US |
