USPP34398P3

USPP34398P3 - Banana plant named ‘QCAV-4’

Info

Publication number: USPP34398P3
Application number: US16/873,740
Authority: US
Inventors: James Dale; Robert Harding; Harjeet Khanna; Anthony James; Jennifer Kleidon; Mark Smith; Upendra Shekhawat
Original assignee: Australian Banana Research Pty Ltd
Current assignee: Queensland University of Technology QUT
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2022-07-05
Anticipated expiration: 2040-06-19
Also published as: US20210400858P1

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 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 10×5 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 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 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 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 - ≤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° 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 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 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 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 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 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 ˜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 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:

>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 <10⁻⁵), 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.

Claims

We claim:

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