Patent Publication Number: US-PP34398-P3

Title: Banana plant named ‘QCAV-4’

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&#39;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 &gt;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: 
     
       
         
           
               
            
               
                 &gt;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*   
               
               
                   
               
               
                 &gt;ORF_156_(frame_3)  
               
               
                 (SEQ ID NO: 2) 
               
               
                 MRFLPEVSACPWHIWSSMNALSCVSMSLAHLVIHECPQP*   
               
               
                   
               
               
                 &gt;ORF_111_(frame_1)  
               
               
                 (SEQ ID NO: 3) 
               
               
                 MHVMLYSWIRRGREDDSGGSIRITHYYGQFKLKAGANSH*   
               
               
                   
               
               
                 &gt;ORF_87_(frame_1)  
               
               
                 (SEQ ID NO: 4) 
               
               
                 MCYSDRSSRVVFPAPPNPTIEHHMHSGIIENKNLKFSTEKCFVIVRRLVH  
               
               
                   
               
               
                 KTENVK* 
               
               
                   
               
               
                 &gt;ORF_71_(frame_3)  
               
               
                 (SEQ ID NO: 5) 
               
               
                 MPLPTVVPKMDPHPRGASWKKKTFQPRLQSKWIDVNMLEQLWRIYCGVNK  
               
               
                   
               
               
                 LTLRQLNNTLRTFLMY*   
               
               
                   
               
               
                 &gt;ORF_49_(frame_3)  
               
               
                 (SEQ ID NO: 6) 
               
               
                 MTKCARDMLTQLRAFMDDQMCQGHADTSGRKRMDSWASDDVQGAEGAEAL  
               
               
                   
               
               
                 QVCRQYLQVWTINDVQNLKRSKVSPLLFLFAGEGYELWTLADL*   
               
               
                   
               
               
                 &gt;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 &lt;10 −5 ), 80-mer sliding window (35% homology with E value &lt;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 .