Banana plant named `QCAV-4`

Abstract

A new banana cultivar `QCAV-4` is provided that, when under significant disease pressure, remains largely free from infection by Fusarium wilt tropical race 4 (TR4). In the absence of such significant disease pressure, `QCAV-4` appears to be essentially phenotypically identical to the wild type parent Cavendish Grand Nain. This includes in relation to immature and mature plant characteristics, fruit characteristics, and yield.

Description

Latin name of the genus and species of the plant claimed: Musa acuminata.

Variety denomination: `QCAV-4`.

BACKGROUND OF THE INVENTION

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.

BRIEF SUMMARY OF THE INVENTION

`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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a digital image of Southern blot analysis of wild type Cavendish Grand Nain (parent) and `QCAV-4`, showing endogenous RGA2 copies and transgenic RGA2 insertions.

FIG. 1B is a digital image of Southern blot analysis of 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` showing distinct pattern of transgenic RGA2 insertions, in addition to the three endogenous RGA2 copies.

FIG. 2A is a schematic diagram showing the general organization of the insert in event `QCAV-4`.

FIG. 2B is a schematic diagram showing the rearrangements in the genome/T-DNA and inter T-DNA junctions of event `QCAV-4`, demonstrating seven (7) new ORFs have been identified in the inter T-DNA regions of the transgenic insert of `QCAV-4`.

FIG. 2C is a schematic diagram showing that there is no evidence of expression of the seven new open reading frames (ORFs) in `QCAV-4`.

FIGS. 3-4 are photographs showing `QCAV-4` at adult stage.

FIG. 5 is photograph showing `QCAV-4` at adult stage and developing fruit.

FIGS. 6-7 are photographs showing `QCAV-4` at adult stage.

FIGS. 8-9 are graphical representations of the data shown in Tables 7-8, respectively.

FIG. 10 is a graphical representation of the data shown in Table 9.

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.

SEQUENCE LISTING

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.

DETAILED DESCRIPTION

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.

BOTANICAL DESCRIPTION

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.

TABLE-US-00001 TABLE 1 Comparison of `QCAV-4` to parent `Cavendish Grand Nain`* `Cavendish Organ/Plant Part: Context `QCAV-4` Grand Nain` Ploidy: triploid triploid Pseudostem: overlapping of weak weak leaf sheaths Pseudostem: tapering absent or weak absent or weak Pseudostem: colour purple purple Pseudostem: anthocyanin medium to strong medium colouration Pseudostem: colour of inner purple purple side of basal sheath Plant: compactness of crown compact compact Plant: growth habit drooping drooping Petiole: attitude of wings at base curved outwards curved outwards Leaf blade: colour of midrib on green green lower side Leaf blade: shape of base both sides acute both sides acute Leaf blade: waxiness on lower medium weak to medium side Leaf blade: width broad broad Leaf blade: glossiness of upper absent absent side Peduncle: diameter large large Peduncle: pubescence present present Peduncle: curvature medium to strong medium to strong Bunch: length long long Bunch: shape cylindrical cylindrical Bunch: attitude of fruits moderately moderately turned up turned up Bunch: compactness medium medium Bunch: number of hands many many Rachis: attitude of male part vertical vertical Rachis: prominence of scars weak weak Rachis: persistence of bracts absent or weak absent or weak Rachis: persistence of present present hermaphrodite flowers Fruit: colour of peel (before greenish yellow greenish yellow maturity) (RHS 141C) (RHS 141C) Fruit: persistence of floral organs present present Male inflorescence: persistence present present Male inflorescence: shape narrow ovate narrow ovate Male inflorescence: opening closed or slightly closed or slightly of bracts open open Bract: colour of inner side orange red orange red Bract: shape of apex broad acute broad acute *For most characteristics, 4-5 individual plants were assessed.

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.times.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`.

TABLE-US-00002 TABLE 2 Resistance of Plants to Fusarium wilt tropical race 4 (TR4). Number Number Percent Variety of Plants Infected Infected `Cavendish Grand Nain` 50 32 64 `Cavendish Williams` 50 38 76 (unpatented) `RGA2-5`(unpatented) 50 14 28 `RGA2-2`(unpatented) 50 8 16 `RGA2-3`(unpatented) 50 3 6 `QCAV-4` 50 0 0

TABLE-US-00003 TABLE 3 Fruit Production Characteristics Plant crop Ratoon 1 No. of No. of Bunch fingers Bunch fingers weight (top Cycle 1 weight (top Cycle 2 Line (kg) hand) (days) (kg) hand) (days) Grand Nain 33.1 24.0 377.3 29.9 20.0 212.4 Williams 30.7 23.0 324.7 29.8 22.2 212.9 RGA2-2 29.1 22.2 268.3 27.6 22.3 223.8 RGA2-3 30.1 24.9 328.9 23.8 20.2 200.8 _QCAV-4 28.1 22.5 331.1 24.3 19.7 199.2 RGA2-5 35.5 24.8 341.6 30.6 22.0 212.9 Ratoon 2 Bunch weight No. of fingers Line (kg) (top hand) Cycle 3 (days) Grand Nain 31.7 26.5 206.6 Williams 40.4 25.9 211.8 RGA2-2 28.1 24.1 215.5 RGA2-3 29.2 26.1 211.7 _QCAV-4 33.7 25.3 206.9 RGA2-5 37.3 29.5 212.8

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.

TABLE-US-00004 TABLE 4 Additional phenotypic details for `QCAV-4`* Characteristic QCAV-4 phenotype Height of the pseudostem 2 - (2.1 to 2.9 m). (mean value = 2.78 m for three plants). Leaf habit/growth habit (upright, 3 - Drooping. spreading, drooping) Pseudostem diameter 83.7 cm (mean value for 3 plants). Attitude of petiole wings at base 1 - Open with margins spreading. (curved outwards, straight, slightly curved inwards, moderately curved inwards, overlapping) Petiole margins 5 - Not winged and not clasping the pseudostem. Petiole wing type 1 - Dry. Edge of petiole margin color 1 - Colourless (without a colour line along) Petiole length 1 - .ltoreq.50 cm. Typically measured at 38 cm. Blotches at the petiole base 3 - Large blotches Color designation of leaf blade Green (137C) midrib on lower side Leaf blade shape of base (both sides 1 - Both sides rounded. rounded, one side rounded and one side acute, both sides acute) Waxiness of lower side of leaf blade 3 - Moderately waxy. Leaf blade length 3 - 221 to 260 cm. Typically measured at 222.7 cm. Leaf blade width 3 - 81 to 90 cm. Typically measured at 90 cm. Leaf blade ratio length/width 5 - 2.4 to 2.6. Typically measured at 2.5. Appearance of leaf lower surface 1 - Dull. Color designation of midrib Green (137C) ventral surface Peduncle length, width, diameter 2 - 31-60 cm 2 - 7-12 cm Empty nodes on peduncle 3-4 Peduncle hairiness 3 - Very hairy, short hairs (similar to velvet touch). Bunch position 1 - Hanging vertically. Bunch shape (cylindrical, irregular, 1 - Cylindrical. conical) Bunch appearance 1 - Lax (one can easily place one's hand between the hands of fruit). Rachis type 2 - Present and male bud may be degenerated or persistent. Rachis position 1 - Falling vertically. Male bud type 1 - Normal (present) Male bud shape 3 - Intermediate. Bract base shape 1 - Small shoulder. Bract apex shape 2 - Slightly pointed. Color of the bract internal face 169A. Prominence of scars on the rachis 2 - Not prominent. (weak, strong) Fading of color on bract base 1 - Color discontinuing towards the base (loss of pigmentation at the base). Male bract shape 3 - Ovate. Typical width of bract Approximately 12 cm Typical length of bract Approximately 28 cm Male bract lifting 3 - Lifting two or more at a time. Bract behavior before falling 1 - Revolute (rolling). Wax on the bract 3 - Moderately waxy. Presence of grooves on the bract 2 - Moderate grooving (parallel ridges are distinguishable). Male flower behavior 4 - Neutral/male flowers persistent. Persistence of bracts on the rachis 3 - Male flowers/bracts above the (absent or weak, strong) male bud (but the stalk is bare above flowers/bracts). Shape of bract apex (narrow acute, 2 - Slightly pointed. broad acute, right angle, obtuse, emarginate) Fruit position 3 - Curved upward (obliquely, at a 45.degree. angle upward). Fruit shape longitudinal curvature 3 - Curved (sharp curve) Fruit longitudinal ridges (absent or 2 - Slightly ridged weak, moderate, strong) Fruit shape of apex 3 - Blunt-tipped (rounded, truncate, bottle-necked, pointed) Fruit persistence of floral organs 2 - Persistent style. Adherence of the fruit peel 1 - Fruit peels easily Cracks in fruit peel 1 - Without cracks Fruit eating quality and main use 1 - Dessert *phenotypic characteristics are presented using the descriptors set out in IPGRI, I. MAD. 1996. Descriptors for Banana. For most, observations from 3-6 individual plants.

TABLE-US-00005 TABLE 5 `QCAV-4` bunch characteristics* BUNCH CHARACTERISTICS DIA- PLANT LENGTH METER ATTITUDE ID (cm) (cm) SHAPE OF FRUIT 20221 88.3 122.6 CONICAL MODERATLY TURNED UP 20223 72.0 123.0 CONICAL MODERATLY TURNED UP 20229 83.0 127.0 CONICAL MODERATLY TURNED UP 20231 75.0 113.0 CONICAL MODERATLY TURNED UP 20241 72.0 120.0 CONICAL MODERATLY TURNED UP 20242 77.5 110.0 CONICAL MODERATLY TURNED UP 20247 76.0 132.0 CONICAL MODERATLY TURNED UP 20248 82.0 142.0 CONICAL MODERATLY TURNED UP 20251 88.0 122.5 CONICAL MODERATLY TURNED UP 20255 59.0 118.0 CONICAL MODERATLY TURNED UP 20257 88.5 122.3 CONICAL MODERATLY TURNED UP 20263 83.0 128.0 CONICAL MODERATLY TURNED UP 20268 80.0 146.0 CONICAL MODERATLY TURNED UP 20269 88.5 126.0 CONICAL MODERATLY TURNED UP 20273 84.0 120.0 CONICAL MODERATLY TURNED UP BUNCH CHARACTERISTICS COM- Ripen- LONGI- PLANT PACT- # OF ing CURVA- TUDINAL LENGTH ID NESS HANDS stage TURE RIDGES (cm) 20221 MEDIUM 11 6 EVENLY MODER- 16.0 CURVED ATE 20223 MEDIUM 8 6 EVENLY MODER- 14.6 CURVED ATE 20229 MEDIUM 10 6 EVENLY MODER- 14.4 CURVED ATE 20231 MEDIUM 10 6 EVENLY MODER- 14.2 CURVED ATE 20241 MEDIUM 6 EVENLY MODER- 13.6 CURVED ATE 20242 MEDIUM 10 6 EVENLY MODER- 14.0 CURVED ATE 20247 MEDIUM 10 6 EVENLY MODER- 13.8 CURVED ATE 20248 MEDIUM 10 6 EVENLY MODER- 15.4 CURVED ATE 20251 MEDIUM 11 6 EVENLY MODER- 15.4 CURVED ATE 20255 MEDIUM 8 6 EVENLY MODER- 13.7 CURVED ATE 20257 MEDIUM 10 6 EVENLY MODER- 14.5 CURVED ATE 20263 MEDIUM 10 6 EVENLY MODER- 13.8 CURVED ATE 20268 MEDIUM 9 6 EVENLY MODER- 15.5 CURVED ATE 20269 MEDIUM 11 6 EVENLY MODER- 14.0 CURVED ATE 20273 MEDIUM 11 6 EVENLY MODER- 13.0 CURVED ATE *Observations from 15 individual plants. Bunch averages:_Length: 79.8 cm; Diameter: 124.8 cm; No. hands: 9.9

TABLE-US-00006 TABLE 6 `QCAV-4` fruit characteristics FRUIT CHARACTERISTICS THICK- LENGTH NESS FRUIT OF OF COLOUR PLANT WIDTH PEDICEL SHAPE OF PEEL OF PEEL ID (cm) (cm) APEX (mm) (RH52015) 20221 3.5 2.8 TRUNCATE 3.8 Light Greenish Yellow 3B- 3D and 4B 20223 3.2 3.4 TRUNCATE 4.1 Light Greenish Yellow 3B- 3D and 4B 20229 3.4 2.9 TRUNCATE 3.2 Light Greenish Yellow 3B- 3D and 4B 20231 3.4 3.0 TRUNCATE 3.2 Light Greenish Yellow 3B- 3D and 4B 20241 3.4 3.2 TRUNCATE 3.3 Light Greenish Yellow 3B- 3D and 4B 20242 3.5 2.6 TRUNCATE 3.0 Light Greenish Yellow 3B- 3D and 4B 20247 3.3 3.4 TRUNCATE 3.5 Light Greenish Yellow 3B- 3D and 4B 20248 3.9 3.3 TRUNCATE 3.5 Light Greenish Yellow 3B- 3D and 4B 20251 3.3 3.3 TRUNCATE 3.7 Light Greenish Yellow 3B- 3D and 4B 20255 3.4 2.2 TRUNCATE 3.2 Light Greenish Yellow 3B- 3D and 4B 20257 3.6 2.5 TRUNCATE 4.0 Light Greenish Yellow 3B- 3D and 4B 20263 3.3 3.2 TRUNCATE 4.5 Light Greenish Yellow 3B- 3D and 4B 3.7 3.2 TRUNCATE 3.1 Light 20268 Yellow 3B- 3D and 4B 20269 3.4 3.8 TRUNCATE 3.2 Light Greenish Yellow 3B- 3D and 4B 20273 2.8 3.1 TRUNCATE 2.7 Light Greenish Yellow 3B- 3D and 4B FRUIT CHARACTERISTICS PLANT ADHERENCE COLOUR OF FIRMNESS PRESENCE ID OF PEEL FLESH OF FLESH OF SEED 20221 PEELS CREAM MEDIUM ABSENT EASILY 20223 PEELS CREAM MEDIUM ABSENT EASILY 20229 PEELS CREAM MEDIUM ABSENT EASILY 20231 PEELS CREAM MEDIUM ABSENT EASILY 20241 PEELS CREAM MEDIUM ABSENT EASILY 20242 PEELS CREAM MEDIUM ABSENT EASILY 20247 PEELS CREAM MEDIUM ABSENT EASILY 20248 PEELS CREAM MEDIUM ABSENT EASILY 20251 PEELS CREAM MEDIUM ABSENT EASILY 20255 PEELS CREAM MEDIUM ABSENT EASILY 20257 PEELS CREAM MEDIUM ABSENT EASILY 20263 PEELS CREAM MEDIUM ABSENT EASILY PEELS CREAM MEDIUM ABSENT EASILY 20269 PEELS CREAM MEDIUM ABSENT EASILY 20273 PEELS CREAM MEDIUM ABSENT EASILY *Observations from 15 individual plants. Fruit averages: _Length: 14.4 cm; Width: 3.4 cm; Peel thickness: 0.35 cm

TABLE-US-00007 TABLE 7 Average cycle time in days per cycle for `QCAV-4` healthy plants only* Cycle time days Plant R1 R2 R3 R4 `RGA2-2` 339.5 223.8 208.1 216 219.4 `RGA2-3` 329 200.8 210.7 201.5 187 `QCAV-4` 331.1 199.2 210.8 174.9 189.8 `RGA2-5` 341.6 212.9 208.6 205.4 207.1 `Grand Nain` 327.3 211.7 206.4 206.2 213.5 `Williams` 324.7 212.3 219.3 206.3 232 *Observations from 50 individual plants for each variety.

TABLE-US-00008 TABLE 8 Average yield in kg per cycle for `QCAV-4` healthy plants only* Average yield Kg Plant R1 R2 R3 R4 `RGA2-2` 29.1 27.6 28 31.1 36.4 `RGA2-3` 30 23.8 27.8 25.4 31.9 `QCAV-4` 28.1 24.3 31.7 28.5 34.8 `RGA2-5` 35.5 30.6 32.3 32 35.8 `Grand 33.1 29.6 32.1 29.8 35.7 Nain` `Williams` 30.7 30.1 35.1 33.7 37.4 *Observations from 50 individual plants for each variety.

TABLE-US-00009 TABLE 9 Comparative TR4 resistance* Cumulative new infections per cycle (%) Plant R1 R2 R3 R4 `RGA2-2` 2 2 2 8 20 `RGA2-3` 2 2 2 2 6 `QCAV-4 0 0 0 0 2 `RGA2-5` 2 6 10 16 36 `Grand Nain` 6 26 40 54 66 `Williams` 2 14 22 38 84 *Observations from 50 individual plants for each variety.

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 FIG. 1A, Southern analysis was consistent with four transgene copies in `QCAV-4`, in addition to the endogenous RGA2 genes. Cavendish Grand Nain is a triploid and it could be expected to have three endogenous copies of RGA2. Two distinct bands were identified in the wild type, indicating that two of three endogenous copies may have migrated together.

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 FIG. 1B, each line has a distinct pattern of transgenic RGA2 insertions, in addition to the three endogenous RGA2 copies.

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 .about.75 Gbp data was obtained in CLR mode (4.9 M reads with a read length N50 of 17,973 bp). This corresponds to .about.42.times. 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 .about.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 FIGS. 2A-2C). In addition, two fragmented portions of the `RGA2` ORF have recombined in opposite directions and inserted between T-DNA2 and T-DNA3. There are two genome/T-DNA and 3 inter T-DNA junctions with various levels of rearrangement (FIGS. 2A-2C).

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:

TABLE-US-00010 >ORF_151_(frame_2) (SEQ ID NO: 1) MWVCVSDDFDVKRITREITEYATNGRFMDLTNLNMLQVNLKEEIRGTTFL LVLDDVWNEDPVKWESLLAPLDAGGRGSVVIVTTQSKKVADVTGTMEPYV LEELTEDDSWSLIESHSFREASCSSTNPRMEEIGRKIAKKISGLPYGATA MGRYLRSKHGESSWREVLETETWEMPPAASDVLSALRRSYDNLPPQLKLC FAFCALFTKGYRFRKDTLIHMWIAQNLIQSTESKRSEDMAEECFDDLVCR FFFRYSWGNYVMNDSVHDLARWVSLDEYFRADEDSPLHISKPIRHLSWCS ERITNVLEDNNTGGDAVNPLSSLRTLLFLGQSEFRSYHLLDRMFRMLSRI RVLDFSNCVIRNLPSSVGNLKHLRYLGLSNTRIQRLPESVTRLCLLQTLL LEGCELCRLPRSMSRLVKLRQLKANPDVIADIAKVGRLIELQELKAYNVD KKKGHGIAELSAMNQLHGDLSIRNLQNVEKTRESRKARLDEKQKLKLLDL RWADGRGAGECDRDRKVLKGLRPHPNLRELSIKYYGGTSSPSWMTDQYLP NMETIRLRSCARLTELPCLGQLHILRHLHIDGMSQVRQINLQFYGTGEVS GFPLLELLNIRRMPSLEEWSEPRRNCCYFPRLHKLLIEDCPRLRNLPSLP PTLEELRISRTGLVDLPGFHGNGDVTTNVSLSSLHVSECRELRSLSEGLL QHNLVALKTAAFTDCDSLEFLPAEGFRTAISLESLIMTNCPLPCSFLLPS SLEHLKLQPCLYPNNNEDSLSTCFENLTSLSFLDIKDCPNLSSFPPGPLC QLSALQHLSLVNCQRLQSIGFQALTSLESLTIQNCPRLTMSHSLVEVNNS SDTGLAFNITRWMRRRTGDDGLMLRHRAQNDSFFGGLLQHLTFLQFLKIC QCPQLVTFTGEEEEKWRNLTSLQILHIVDCPNLEVLPANLQSLCSLSTLY IVRCPRIHAFPPGGVSMSLAHLVIHECPQLCQHVPGTFGHP* >ORF_156_(frame_3) (SEQ ID NO: 2) MRFLPEVSACPWHIWSSMNALSCVSMSLAHLVIHECPQP* >ORF_111_(frame_1) (SEQ ID NO: 3) MHVMLYSWIRRGREDDSGGSIRITHYYGQFKLKAGANSH* >ORF_87_(frame_1) (SEQ ID NO: 4) MCYSDRSSRVVFPAPPNPTIEHHMHSGIIENKNLKFSTEKCFVIVRRLVH KTENVK* >ORF_71_(frame_3) (SEQ ID NO: 5) MPLPTVVPKMDPHPRGASWKKKTFQPRLQSKWIDVNMLEQLWRIYCGVNK LTLRQLNNTLRTFLMY* >ORF_49_(frame_3) (SEQ ID NO: 6) MTKCARDMLTQLRAFMDDQMCQGHADTSGRKRMDSWASDDVQGAEGAEAL QVCRQYLQVWTINDVQNLKRSKVSPLLFLFAGEGYELWTLADL* >ORF_7_(frame_2) (SEQ ID NO: 7) MDRHLKSRIRFWFKQQWPRQLNNTLRCKQIDA*

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.sup.-5), 80-mer sliding window (35% homology with E value <10.sup.-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.times.10.sup.-4.

SEQUENCE LISTINGS

1

71991PRTArtificial SequenceORF 151 1Met Trp Val Cys Val Ser Asp Asp Phe Asp Val Lys Arg Ile Thr Arg1 5 10 15Glu Ile Thr Glu Tyr Ala Thr Asn Gly Arg Phe Met Asp Leu Thr Asn 20 25 30Leu Asn Met Leu Gln Val Asn Leu Lys Glu Glu Ile Arg Gly Thr Thr 35 40 45Phe Leu Leu Val Leu Asp Asp Val Trp Asn Glu Asp Pro Val Lys Trp 50 55 60Glu Ser Leu Leu Ala Pro Leu Asp Ala Gly Gly Arg Gly Ser Val Val65 70 75 80Ile Val Thr Thr Gln Ser Lys Lys Val Ala Asp Val Thr Gly Thr Met 85 90 95Glu Pro Tyr Val Leu Glu Glu Leu Thr Glu Asp Asp Ser Trp Ser Leu 100 105 110Ile Glu Ser His Ser Phe Arg Glu Ala Ser Cys Ser Ser Thr Asn Pro 115 120 125Arg Met Glu Glu Ile Gly Arg Lys Ile Ala Lys Lys Ile Ser Gly Leu 130 135 140Pro Tyr Gly Ala Thr Ala Met Gly Arg Tyr Leu Arg Ser Lys His Gly145 150 155 160Glu Ser Ser Trp Arg Glu Val Leu Glu Thr Glu Thr Trp Glu Met Pro 165 170 175Pro Ala Ala Ser Asp Val Leu Ser Ala Leu Arg Arg Ser Tyr Asp Asn 180 185 190Leu Pro Pro Gln Leu Lys Leu Cys Phe Ala Phe Cys Ala Leu Phe Thr 195 200 205Lys Gly Tyr Arg Phe Arg Lys Asp Thr Leu Ile His Met Trp Ile Ala 210 215 220Gln Asn Leu Ile Gln Ser Thr Glu Ser Lys Arg Ser Glu Asp Met Ala225 230 235 240Glu Glu Cys Phe Asp Asp Leu Val Cys Arg Phe Phe Phe Arg Tyr Ser 245 250 255Trp Gly Asn Tyr Val Met Asn Asp Ser Val His Asp Leu Ala Arg Trp 260 265 270Val Ser Leu Asp Glu Tyr Phe Arg Ala Asp Glu Asp Ser Pro Leu His 275 280 285Ile Ser Lys Pro Ile Arg His Leu Ser Trp Cys Ser Glu Arg Ile Thr 290 295 300Asn Val Leu Glu Asp Asn Asn Thr Gly Gly Asp Ala Val Asn Pro Leu305 310 315 320Ser Ser Leu Arg Thr Leu Leu Phe Leu Gly Gln Ser Glu Phe Arg Ser 325 330 335Tyr His Leu Leu Asp Arg Met Phe Arg Met Leu Ser Arg Ile Arg Val 340 345 350Leu Asp Phe Ser Asn Cys Val Ile Arg Asn Leu Pro Ser Ser Val Gly 355 360 365Asn Leu Lys His Leu Arg Tyr Leu Gly Leu Ser Asn Thr Arg Ile Gln 370 375 380Arg Leu Pro Glu Ser Val Thr Arg Leu Cys Leu Leu Gln Thr Leu Leu385 390 395 400Leu Glu Gly Cys Glu Leu Cys Arg Leu Pro Arg Ser Met Ser Arg Leu 405 410 415Val Lys Leu Arg Gln Leu Lys Ala Asn Pro Asp Val Ile Ala Asp Ile 420 425 430Ala Lys Val Gly Arg Leu Ile Glu Leu Gln Glu Leu Lys Ala Tyr Asn 435 440 445Val Asp Lys Lys Lys Gly His Gly Ile Ala Glu Leu Ser Ala Met Asn 450 455 460Gln Leu His Gly Asp Leu Ser Ile Arg Asn Leu Gln Asn Val Glu Lys465 470 475 480Thr Arg Glu Ser Arg Lys Ala Arg Leu Asp Glu Lys Gln Lys Leu Lys 485 490 495Leu Leu Asp Leu Arg Trp Ala Asp Gly Arg Gly Ala Gly Glu Cys Asp 500 505 510Arg Asp Arg Lys Val Leu Lys Gly Leu Arg Pro His Pro Asn Leu Arg 515 520 525Glu Leu Ser Ile Lys Tyr Tyr Gly Gly Thr Ser Ser Pro Ser Trp Met 530 535 540Thr Asp Gln Tyr Leu Pro Asn Met Glu Thr Ile Arg Leu Arg Ser Cys545 550 555 560Ala Arg Leu Thr Glu Leu Pro Cys Leu Gly Gln Leu His Ile Leu Arg 565 570 575His Leu His Ile Asp Gly Met Ser Gln Val Arg Gln Ile Asn Leu Gln 580 585 590Phe Tyr Gly Thr Gly Glu Val Ser Gly Phe Pro Leu Leu Glu Leu Leu 595 600 605Asn Ile Arg Arg Met Pro Ser Leu Glu Glu Trp Ser Glu Pro Arg Arg 610 615 620Asn Cys Cys Tyr Phe Pro Arg Leu His Lys Leu Leu Ile Glu Asp Cys625 630 635 640Pro Arg Leu Arg Asn Leu Pro Ser Leu Pro Pro Thr Leu Glu Glu Leu 645 650 655Arg Ile Ser Arg Thr Gly Leu Val Asp Leu Pro Gly Phe His Gly Asn 660 665 670Gly Asp Val Thr Thr Asn Val Ser Leu Ser Ser Leu His Val Ser Glu 675 680 685Cys Arg Glu Leu Arg Ser Leu Ser Glu Gly Leu Leu Gln His Asn Leu 690 695 700Val Ala Leu Lys Thr Ala Ala Phe Thr Asp Cys Asp Ser Leu Glu Phe705 710 715 720Leu Pro Ala Glu Gly Phe Arg Thr Ala Ile Ser Leu Glu Ser Leu Ile 725 730 735Met Thr Asn Cys Pro Leu Pro Cys Ser Phe Leu Leu Pro Ser Ser Leu 740 745 750Glu His Leu Lys Leu Gln Pro Cys Leu Tyr Pro Asn Asn Asn Glu Asp 755 760 765Ser Leu Ser Thr Cys Phe Glu Asn Leu Thr Ser Leu Ser Phe Leu Asp 770 775 780Ile Lys Asp Cys Pro Asn Leu Ser Ser Phe Pro Pro Gly Pro Leu Cys785 790 795 800Gln Leu Ser Ala Leu Gln His Leu Ser Leu Val Asn Cys Gln Arg Leu 805 810 815Gln Ser Ile Gly Phe Gln Ala Leu Thr Ser Leu Glu Ser Leu Thr Ile 820 825 830Gln Asn Cys Pro Arg Leu Thr Met Ser His Ser Leu Val Glu Val Asn 835 840 845Asn Ser Ser Asp Thr Gly Leu Ala Phe Asn Ile Thr Arg Trp Met Arg 850 855 860Arg Arg Thr Gly Asp Asp Gly Leu Met Leu Arg His Arg Ala Gln Asn865 870 875 880Asp Ser Phe Phe Gly Gly Leu Leu Gln His Leu Thr Phe Leu Gln Phe 885 890 895Leu Lys Ile Cys Gln Cys Pro Gln Leu Val Thr Phe Thr Gly Glu Glu 900 905 910Glu Glu Lys Trp Arg Asn Leu Thr Ser Leu Gln Ile Leu His Ile Val 915 920 925Asp Cys Pro Asn Leu Glu Val Leu Pro Ala Asn Leu Gln Ser Leu Cys 930 935 940Ser Leu Ser Thr Leu Tyr Ile Val Arg Cys Pro Arg Ile His Ala Phe945 950 955 960Pro Pro Gly Gly Val Ser Met Ser Leu Ala His Leu Val Ile His Glu 965 970 975Cys Pro Gln Leu Cys Gln His Val Pro Gly Thr Phe Gly His Pro 980 985 990239PRTArtificial SequenceORF 156 2Met Arg Phe Leu Pro Glu Val Ser Ala Cys Pro Trp His Ile Trp Ser1 5 10 15Ser Met Asn Ala Leu Ser Cys Val Ser Met Ser Leu Ala His Leu Val 20 25 30Ile His Glu Cys Pro Gln Pro 35339PRTArtificial SequenceORF 111 3Met His Val Met Leu Tyr Ser Trp Ile Arg Arg Gly Arg Glu Asp Asp1 5 10 15Ser Gly Gly Ser Ile Arg Ile Thr His Tyr Tyr Gly Gln Phe Lys Leu 20 25 30Lys Ala Gly Ala Asn Ser His 35456PRTArtificial SequenceORF 87 4Met Cys Tyr Ser Asp Arg Ser Ser Arg Val Val Phe Pro Ala Pro Pro1 5 10 15Asn Pro Thr Ile Glu His His Met His Ser Gly Ile Ile Glu Asn Lys 20 25 30Asn Leu Lys Phe Ser Thr Glu Lys Cys Phe Val Ile Val Arg Arg Leu 35 40 45Val His Lys Thr Glu Asn Val Lys 50 55566PRTArtificial SequenceORF 71 5Met Pro Leu Pro Thr Val Val Pro Lys Met Asp Pro His Pro Arg Gly1 5 10 15Ala Ser Trp Lys Lys Lys Thr Phe Gln Pro Arg Leu Gln Ser Lys Trp 20 25 30Ile Asp Val Asn Met Leu Glu Gln Leu Trp Arg Ile Tyr Cys Gly Val 35 40 45Asn Lys Leu Thr Leu Arg Gln Leu Asn Asn Thr Leu Arg Thr Phe Leu 50 55 60Met Tyr65693PRTArtificial SequenceORF 49 6Met Thr Lys Cys Ala Arg Asp Met Leu Thr Gln Leu Arg Ala Phe Met1 5 10 15Asp Asp Gln Met Cys Gln Gly His Ala Asp Thr Ser Gly Arg Lys Arg 20 25 30Met Asp Ser Trp Ala Ser Asp Asp Val Gln Gly Ala Glu Gly Ala Glu 35 40 45Ala Leu Gln Val Cys Arg Gln Tyr Leu Gln Val Trp Thr Ile Asn Asp 50 55 60Val Gln Asn Leu Lys Arg Ser Lys Val Ser Pro Leu Leu Phe Leu Phe65 70 75 80Ala Gly Glu Gly Tyr Glu Leu Trp Thr Leu Ala Asp Leu 85 90732PRTArtificial SequenceORF 7 7Met Asp Arg His Leu Lys Ser Arg Ile Arg Phe Trp Phe Lys Gln Gln1 5 10 15Trp Pro Arg Gln Leu Asn Asn Thr Leu Arg Cys Lys Gln Ile Asp Ala 20 25 30

Claims

We claim:

1. A new and distinct variety of banana plant, substantially as herein shown and described.