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.
