Method for determining the level of zygosity of a seed
11668703 · 2023-06-06
Assignee
Inventors
Cpc classification
G01N21/6486
PHYSICS
International classification
G01N33/50
PHYSICS
G01N1/28
PHYSICS
Abstract
The invention relates to a method for determining the level of transgene zygosity in a poaceae seed, the transgene being genetically linked to a gene coding for a fluorescent protein (FP protein) under the control of a promoter operative in the endosperm, comprising the step of exposing the endosperm of the seed to a wavelength exciting the FP protein, and measuring the intensity of the emitted fluorescence.
Claims
1. A method for determining a level of transgene zygosity in a poaceae seed, the method comprising: providing a poaceae seed comprising a transgene that is genetically linked to a gene coding for a fluorescent protein (FP protein) under the control of a promoter operative in endosperm of the seed; exposing the endosperm of the seed to a wavelength exciting the FP protein; and measuring intensity of fluorescence emitted by the endosperm, wherein the intensity of fluorescence emitted by the endosperm of the seed is further compared to intensity of fluorescence measured for individual seeds from a batch of seeds, wherein the batch of seeds is from the same plant or from the same transgenic event as the seed, wherein four classes (0×, 1×, 2×or 3×) of transgene zygosity levels are determined based on relative fluorescence intensity of the seeds of the batch.
2. The method of claim 1, wherein the intensity of fluorescence emitted by the endosperm of the seed is calculated after processing an image of the seed in a computer.
3. The method of claim 1, wherein the promoter is selected from the group consisting of HMWG promoter, maize gamma Zein promoter, CaMV35S promoter, rice actin promoter, maize polyubiquitin promoter, rice tubulin promoter, and CsVMV promoter.
4. The method of claim 1, wherein the seed has been soaked before being exposed to the wavelength exciting the FP protein.
5. The method of claim 4, wherein the soaking has been performed for a duration between 4 and 12 hours.
6. The method of claim 1, further comprising performing a PCR to validate ploidy of the transgene.
7. The method of claim 1, wherein the seed is a maize or a wheat seed.
8. A method for reading fluorescence emitted by transgenic poaceae seeds, the method comprising: a) providing a poaceae seed comprising a transgene that is genetically linked to a gene coding for a fluorescent protein (FP protein) under the control of a promoter operative in endosperm of the seed; b) dispatching seeds from a seed lot into wells of a plate; c) placing the plate on a device and exposing the plate to an excitation wavelength; d) measuring fluorescence emitted by each seed upon excitation; and e) analyzing fluorescence emitted by each seed with a statistic tool to create four classes according to relative fluorescence intensity of the seeds.
9. A method for reading fluorescence emitted by transgenic poaceae seeds, the method comprising: a) providing a poaceae seed comprising a transgene that is genetically linked to a gene coding for a fluorescent protein (FP protein) under the control of a promoter operative in endosperm of the seed; b) dispatching individual seeds onto a belt moving through a device and exposing the seeds on the belt to an excitation wavelength; c) measuring the fluorescence emitted by each seed upon excitation; and d) analyzing the fluorescence emitted by each seed with a statistic tool to create four classes according to relative fluorescence intensity of the seeds.
10. A method for sorting a transgenic poaceae seed, the method comprising: a) providing a poaceae seed comprising a transgene that is genetically linked to a gene coding for a fluorescent protein (FP protein) under the control of a promoter operative in endosperm of the seed; b) exposing the endosperm of the seed to a wavelength exciting the FP protein; c) measuring fluorescence emitted by each seed upon excitation; d) allocating a measurement of the fluorescence emitted by each seed to one of four classes of fluorescence intensity, as determined in claim 8; and e) sorting the seed according to its allocated class of fluorescence intensity.
11. A method for sorting a transgenic poaceae seed, the method comprising: a) providing a poaceae seed comprising a transgene that is genetically linked to a gene coding for a fluorescent protein (FP protein) under the control of a promoter operative in endosperm of the seed; b) exposing the endosperm of the seed to a wavelength exciting the FP protein; c) measuring fluorescence emitted by each seed upon excitation; d) allocating a measurement of the fluorescence emitted by each seed to one of four classes of fluorescence intensity, as determined in claim 9; and e) sorting the seed according to its allocated class of fluorescence intensity.
12. The method of claim 6, wherein the PCR is qPCR.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1. Application to Maize
(10) 1-1—Genetic Modified Maize.
(11) The seeds come from plants genetically transformed with Agrobacterium tumefaciens (Ishida et al., 1996), each construct (T-DNA) comprising: One or more Genes Of Interest (GOI) under the control of suitable promoter; A marker (selection) gene, under the control of a constitutive promoter, used to select the plants having integrated the transgene, such as an herbicide resistance gene, in particular the bar gene that confers resistance to Glufosinate, phosphinotricin and Bialaphos. A reporter gene to obtain fluorescence in the endosperm (a ZsGreen encoding gene, as described above (SEQ ID NO: 3).), under the control of a promoter functional in the endosperm, in particular the HMWG promoter (SEQ ID NO: 2).
(12) For the experiments the seed lots used come from a self-fertilization of regenerated transgenic plants. The transformation events have a molecular profile with one intact copy of the T-DNA. Genetically, there are 3 different genotypes in each seed lot with the following proportions: ¼ of wild grains (WT) or null segregant, ½ of heterozygous (HE) grains and ¼ of homozygous (HO) grains. Due to the double fertilization, the albumen of the grain can exist with four different number of transgenes: the genotype WT with no transgene, HE with 1 copy (1×) of the transgene when originating from the paternal zygote or 2 copies (2×) of the transgene when originating from the maternal zygote and 3 copies (3×) for the genotype HO with one 1 paternal and 2 maternal copies.
(13) 1-2—Visual Classification of ZsGreen Seeds.
(14) 96 lots of seeds were visually sorted by 3 different operators. The observations were made with a Leica MZ10 F fluorescence microscope equipped with the Leica EL6000 external light source. A magnification ×8 and excitation with a wavelength of 480 nm and a stopping filter at 510 nm were used (kit fluorescence GFP Plus).
(15) The grains expressing the ZsGreen were separated into 2 fractions (1 low intensity fraction and 1 high intensity fraction).
(16) For each of the 96 batches, 3 kernels from the high intensity FP fraction were sown and genotyping by quantitative PCR were done on corresponding plantlets. The results gave a rate of 63% of homozygous plantlets and 37% of hemizygous plantlets. This experiment shows that this visual sorting of seeds by the intensity level of the endosperm FP allows the identification of potentially 3× transgene copy endosperm. An average rate of 63% of 3× endosperm is contained in this high intensity FP fraction while only 33% of FP positive seeds contains 3× copies. This classification has been shown to be similar with the 3 different operators.
(17) 1-3—Classification of ZsGreen Seeds by Image J
(18) 48 maize caryopses are placed in wells of plates “NUNC 48” (THERMOSCIENTIFIC, Waltham, Mass., USA). The seeds are preferably stuck on the bottom of the wells to prevent seed movement, endosperm is on the upper face.
(19) For observation of the fluorescence and acquisition of photos, the Leica MZ10 F modular stereo microscope is used with the Leica EL6000 external light source. High-definition pictures are taken with the Leica DFC420 C, a digital microscope camera with c-mount and with a 5 Mpixel CCD sensor.
(20) The filters kits installed on the microscope are: the kit Fluorescence GFP Plus (emission filter wavelength 480/40 nm; stop filter wavelength 510 nm) the kit fluorescence GFP Plants (emission filter wavelength 470/40 nm; stop filter wavelength 525/50 nm) and the filter nGreen (emission filter wavelength 490/20 nm; stop filter wavelength 530/20 nm), which is the one preferred and further used.
(21) The camera is driven by the Leica Application Suite V3.3.0. This software makes it possible to adjust the exposition of the sensor to fluorescence in order to avoid saturation in the green light.
(22) The pictures are stored for further analysis, with the suite Image J V 1.47, Open Source software available at http://imagej.updatestar.com. Using the function “Measure RGB” the average Green pixels intensity is determined on a 300×300 pixel area of the seed picture. These values are sorted in ascending order and entered in a histogram. Seeds are allocated into four class relative to their relative fluorescence intensity by a Kmeans function (see 2.6). The results for 48 seeds are illustrated in
(23) 1-4—Classification of ZsGreen Seeds by Fluorimeter (Fluostar)
(24) The same plates were used with a fluorimeter.
(25) The FLUOstar OPTIMA Microplate Reader (BMG LABTECH, Ortenberg, Germany) is used to measure fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. This automat is controlled with the OPTIMA Control Software V2.20. Once measured, the data obtained are processed with the suite OPTIMA MARS Data Analysis V2.41. The automat is programed in order to avoid obtaining data with a maximum of 65 000 RFU (Relative Fluorescence Units).
(26) Each well of the plate was scanned by 80 consecutive flashes (SCAN protocol) distributed over a disk 10 mm in diameter centered on each well. An average of 80 values for each well is done. These values are sorted in ascending order and entered in a histogram. The results for 48 seeds are illustrated in
(27) This diagram shows that the fluorescence intensity of the grains does not increase continuously. It is possible to distinguish four levels by a Kmeans function (see 2.6).
(28) 1-5—Comparison of the Two Methods of Fluorescence Reading Image J and FLUOStar
(29) Correlation between fluorescence measurements obtained by Image J and FLUOstar method for the 48 seeds are shown in
(30) The R.sup.2 (coefficient of determination) between the results obtained with the FLUOStar and the Image J software is of R.sup.2=0.9628, indicating that any of the two tested fluorescence analysis software can be used with the same rate of accuracy.
(31) 1-6—Optimization of the Use of FLUOStar
(32) The FLUOstar proposes the “orbital averaging” measurement mode (FAST protocol), which measures the fluorescence emitted on the circumference of a circle centered on the well, the diameter of which having been pre-selected, and the maximum number of flashes depending on the selected diameter
(33) To identify the FAST protocol that is gives the best correlation with to the SCAN protocol (previous example), several diameters and number of flashes are tested (Ø 3 mm, 5 flashes); (Ø 3 mm, 3 flashes); (Ø 3 mm, 10 flashes); (Ø 4 mm, 5 flashes); (Ø 5 mm, 8 flashes); (Ø 5 mm, 12 flashes). These protocols require a measurement time of 30 to 40 seconds per plate.
(34) The number of flashes per well has less influence, especially when the diameter is small (3 mm).
(35) The protocol with Ø 5 mm and 12 flashes was selected, and gives similar results to the SCAN, in a shorter lapse of measure (4 times faster).
(36) 1-7—qPCR Validation of Zygoty Status Identified by Image J or the FLUOstar Method.
(37) With the FLUOstar measurements the grains can be classified according to their fluorescence to predict their zygosity level with regards to the fluorescent protein and hence the transgene. To evaluate the reliability of this test, the results were confirmed by qPCR analysis on the plantlets germinated from these seeds.
(38) Unlike caryopsis whose endosperm is triploid, the plantlets are diploid. It is therefore impossible to verify whether the endosperms are 1× or 2×, the two classes will leads to hemizygous plantlets with the molecular analysis.
(39) For 4 independent seed lots, event 1 to 4, all 48 seeds measured by FLUOstar and classified as wild type (null segregant), homozygous for the transgene (3×) and heterozygous for the transgene (1× or 2×) were sown. The corresponding plantlets were analyzed by qPCR and classified into wild type, homozygous or heterozygous for the transgene. The table below shows the percentage of seeds with identical results between both methods.
(40) TABLE-US-00001 TABLE 1 result concordance of FLUOstar classification on seeds and pPCR on resulting plantlets in percentage. Overall Null segregant Heterozygous Homozygous concordance Event 1 100 94 67 88 Event 2 100 85 86 89 Event 3 100 100 90 97 Event 4 100 96 60 94 total 100 93 76 92
(41) A good correlation is obtained between the qPCR and FLUOstar results. Analysis by fluorimetry for the determination the zygosity on grains, gives better results than visual sorting (92% versus 63%).
(42) 1.8—Use of the Method for Phenotypic Evaluation of Maize Transgenic Events.
(43) A construct containing a gene coding for a fluorescent protein under the control of an endosperm specific promoter, a selection marker and a gene of interest under the control of a suitable promoter is used for genetic transformation. Maize transgenic events were produced in tissue culture and seeds from the transgenic T0 plants harvested. After self-pollination of the T1 plants the segregation rate obtained from the seeds (with regards to the transgene) is 50% of heterozygous seeds, 25% homozygous and 25% wild type.
(44) Submitting the seeds to appropriate light emission and measuring the emitted fluorescence with the FLUOStar method for example made possible to sort of seeds in four categories according to their endosperm zygosity level for the FP: no FP for the 25% wild type seeds. 1× of 2× in the endosperm for the 50% heterozygous seeds for the transgene. 3× for the 25% homozygous seeds for the transgene.
(45) Only seeds from the last category are recovered and used in future development (sown and crossed with another variety to produce a hybrid progeny in order to perform phenotypic evaluation on the progeny). If a lot containing 48 seeds is analyzed, there are generally enough seeds in the last category (3×) to perform the selection scheme. This method is less time consuming than the regular step of sowing numerous seeds followed by sampling/screening of plantlets. The cost of zygosity determination is thus significantly reduced, the time necessary for sampling and doing the molecular analysis (qPCR) is saved as is the cost of the reagents (
(46) An experiment was performed on 288 independent events. For each event, 48 seeds were analyzed. From these 48 seeds of each event, the five seeds having the most important fluorescence value have been retained (from seed 1 to seed 5 by decreasing fluorescence level), sowed and the homozygosity of the plant was checked: on seedlings by Q-PCR (quantitative PCR; it is to be noted that this method gives non-conclusive results in 1% to 2.4% of cases). Or on the ears obtained from the plants grown from these seeds. If the initial seed is homozygous for the GFP, all seeds on the ear will be GFP positive; if the seed is heterozygous, the ear will be segregating (some seeds will be GFP positive and others no)
(47) After analysis of the plants obtained from the sowed seeds, the result is as in Table 2. The success rate corresponds to the number of plants (issued from seed 1 to 5 for each of the 288 independent events) for which the grown plants were effectively homozygous.
(48) TABLE-US-00002 TABLE 2 Percentage of homozygous plants, from all plants grown from the 5 seeds selected with the highest fluorescence level Success rate Seed 1 92.7% Seed 2 92.4% Seed 3 90.1% Seed 4 88.1% Seed 5 86.2%
(49) This table shows that the method makes it possible to select seeds with homozygous genotype with a very high level of success, and makes it possible to save a lot of time and resources.
(50) In a routine protocol, only the two first seeds (seed with the highest detected fluorescence) can be selected as illustrated in
Example 2. Application to Wheat
(51) 2-1—Genetic Modified Wheat.
(52) The seeds are obtained from plants genetically transformed with Agrobacterium tumefaciens (Ishida et al., 2015), each construct comprising: One or more Genes Of Interest (GOI) under the control of suitable promoter; A marker (selection) gene, under the control of a constitutive promoter, used to select the plants having integrated the transgene, such as an herbicide resistance gene, in particular the bar gene that confers resistance to Glufosinate, phosphinotricin, Bialaphos or the nptll selection marker conferring resistance to phosphinotricine. A reporter gene that allows fluorescence in the endosperm (a ZsGreen-encoding gene (SEQ ID NO: 3), under the control of a promoter functional in the endosperm, in particular the HMWG promoter (SEQ ID No: 2).
(53) For the experiments the seed lots were obtained from a self-pollination of regenerated T0 plants.
(54) 2-2—Visual Classification of ZsGreen Seeds
(55) 20 lots of seeds were visually sorted. With the protocol described in example 1.2 and the nGreen filter.
(56) Different levels of intensity were observed for seeds expressing ZsGreen. It was possible to separate the grains expressing ZsGreen from those not expressing ZsGreen. However they were sometimes difficult to differentiate due to endogenous background fluorescence in the seed coat.
(57) After visual sorting and separation, grains were counted to perform a calculation of segregation. The data obtained is given in the following table:
(58) TABLE-US-00003 TABLE 3 twenty seeds lots from independent transformation events sorted according the presence or absence of a fluorescent protein (FP). Events (seed Number of Percentage of seeds Percentage of seeds lots) seeds positive for FP negative for FP Event 1 64 75% 25% Event 2 53 72% 28% Event 3 41 76% 24% Event 4 63 60% 40% Event 5 189 71% 29% Event 6 115 78% 22% Event 7 99 77% 23% Event 8 43 79% 21% Event 9 235 77% 23% Event 10 348 64% 36% Event 11 116 82% 18% Event 12 64 77% 23% Event 13 109 74% 26% Event 14 84 75% 25% Event 15 100 73% 27% Event 16 75 71% 29% Event 17 32 56% 44% Event 18 37 62% 38% Event 19 269 62% 38% Event 20 94 50% 27%
(59) The expected Mendelian segregation is ¾ FP+, ¼ FP− is obtained for most of the lots. It appears that it is possible to discriminate fluorescence-positive from fluorescence-negative grains, with sometimes hesitation to differentiate a wild-type seed from a weakly positive seed.
(60) This observation also reveals a difference of intensity emitted by the fluorescence-positive grains, which seems to demonstrate that the fluorescence intensity may indicate the level of zygosity of the grain.
(61) The seeds of each event were classified after visual evaluation in one of the following three classes: Highly fluorescent grains (FP++) Fluorescent grains (FP+), not considered highly fluorescent Non-fluorescent grains (FP−)
(62) TABLE-US-00004 TABLE 4 sorting of seeds for the event 20, into tree classes, absence of fluorescent protein, and presence of fluorescent protein with two level of intensity. Event Nb seeds FP− FP+ FP++ Event 20 94 25 (27%) 47 (50%) 22 (23%)
(63) The proportions obtained confirm the Mendelian segregation of ¼ seeds homozygous (FP++), ¼ wild-type seeds (FP−) and ½ hemizygous seeds (FP+).
(64) These observations indicate that the ZsGreen protein can be used as a marker of zygosity of the seed.
(65) To improve the sorting, the seeds were imbibed in water at room temperature for a duration of 8 hours. After soaking, the seeds expressing ZsGreen and those not expressing ZsGreen, were more easily sorted visually (
(66) 2-3—Classification of ZsGreen Seeds by Fluorimeter (FLUOstar)
(67) 48 wheat caryopses from a seed lot were placed in wells of plates “NUNC 48” (THERMOSCIENTIFIC, Waltham, Mass., USA). The seeds were preferably stuck on the bottom of the wells to prevent seed movement. The round part of the endosperm was preferably placed facing upwards with the groove stuck to the bottom of plate.
(68) FLUOstar OPTIMA Microplate Reader (BMG LABTECH, Ortenberg, Germany) was used to measure fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. This automat is controlled with the OPTIMA Control Software V2.20. Once measured, the data obtained were processed with the suite OPTIMA MARS Data Analysis V2.41.
(69) The automat was programed in order to avoid obtaining data with a maximum of 65 000 RFU (Relative Fluorescence Units).
(70) Each well of the plate was scanned by 10 consecutive flashes distributed over a disk 2 mm in diameter centered on each well. An average of 10 values for each well is done. These values are sorted in ascending order and entered in a histogram.
(71) The measurements were made on dry seeds and then on imbibed seeds. The results for 48 seeds are illustrated in
(72) 2.4 Soaking the Seeds
(73) Visually, the soaked grains appear swelled, and the differences in fluorescence intensity seem easier to determine, in particular improves the detection of the negative (wild-type) grains, as it seems easier to visually detect positive grains. Fluorescence measurements were made on dry grains and on soaked grains. All events were assessed with the FLUOstar as dry grains and soaked seeds to obtain the two intensity values. All events were soaked directly in the microplate for 8 hours before another run with FLUOstar.
(74) Soaking leads to lower overall intensity values, with a curve having a similar pattern than for dry grains (see
(75) Soaking the seeds provides better separation between classes, especially at the transition classes WT (wild-type) and 1× (hemizygous). Previously soaked wild grains seem to lose any fluorescent intensity, whereas there is a slight endogenous fluorescence in dry wild-type seeds.
(76) 2.5 Seed Orientation for Fluorimeter (FLUOstar) Measurement
(77) For maize, the orientation of the seed is important (opposite side to the embryo that is measured). For wheat, the embryo representing a small part of the grain, the measurement can be made on any side yielding similar results.
(78) 2-6 Use of the Kmeans Function
(79) The fluorescence intensity data in the Excel file are then exported to the R software to perform the Kmeans function.
(80) This tool generates a matrix of each microplate with the 48 wells scanned by the FLUOstar and a graph of these 48 wells according to the increasing intensity of fluorescence.
(81) The Kmeans function also allows dividing the data set in a number of classes defined by assessing the distance of one point to the average of the other points in its class.
(82) The software was parametered as to provide 4 classes that are expected for the four zygosity levels (WT/1×/2×/3×). The classes are represented by different symbols (
(83) The Kmeans function also provides the identity of the four grains having the highest fluorescence intensity.
(84) 2. 7 qPCR Validation of Zygosity Status Identified by the Fluorimeter Method
(85) With the FLUOstar measurements the grains can be classified according to their fluorescence to predict their zygosity level. To evaluate the reliability of this test the results were confirmed by qPCR analysis on the plantlets germinated from these seeds.
(86) Unlike caryopsis whose albumen is triploid, the plantlets are diploid. It is therefore impossible to verify whether the grains are 1× or 2×, the two classes will be detected hemizygous with the molecular analysis.
(87) In a first experiment dry seeds were analyzed: For 4 independent events, 48 seeds were measured by FLUOstar and then sown. The germinated seeds were sampled and analyzed by qPCR. The table below shows the percentage of seeds with identical results between both methods per event:
(88) TABLE-US-00005 TABLE 5 result of concordance of FLUOstar classification of dry seeds and pPCR for the corresponding plantlets. Overall Null segregant Heterozygous Homozygous concordance (%) (%) (%) (%) Event 3 57 73 33.5 58 Event 6 87.5 71.5 44.5 68 Event 10 100 84 70 85 Event 13 91.5 82.6 60 80 total 87 78.5 52.5 75
(89) In a second experiment, imbibed seeds were analyzed. For 2 events, 48 seeds were soaked in water at room temperature for a duration of 8 hours before the fluorescence was measured by FLUOstar and then sown. The table below shows the percentage of seeds with identical results between both methods per event:
(90) TABLE-US-00006 TABLE 6 result of concordance of FLUOstar classification on imbibed seeds and pPCR for the corresponding plantlets. Overall Null segregant Heterozygous Homozygous concordance (%) (%) (%) (%) Event 10 100 100 90 98 Event 13 100 95.5 50 87 total 100 98 70 92
(91) The comparison of the reliability of the results obtained on the dry seeds and the soaked seeds shows that both techniques give good results. However, the measurement on imbibed seeds is more preferred.
(92) 2.8 Comparison of the Method Herein Disclosed and the Method Usually Performed for Detecting Homozygous Seeds (qPCR on Leaf Samples or Half-Seeds)
(93) The method as disclosed above makes it possible to identify homozygous seeds in half the time needed using the prior methods, for a much lower cost (essentially null) where the other qPCR methods have, at least, the cost of reagents.
(94) 2.9 Use of the Method for Phenotypic Evaluation of Transgenic Events.
(95) The method can be used in the same way as described for maize for the phenotypic evaluation of transgenic events in wheat. On a larger scale experiment of 135 events, homozygous plants were recovered for 127 events: success rate is of 94.1%.
(96) V. Conclusion
(97) In conclusion, this demonstrates that one can use the fluorescent reporter genes widely used in biotechnology to detect the state of zygosity of a transgene in a plant breeding system, directly on seeds, by detection of a fluorescence of a specific part of the seeds that is different from the embryo (detection in the endosperm).
(98) The method is also very applicable to angiosperm plants (as herein exemplified on maize and wheat) and also allows determine whether the transgene, in hemizygous plants, comes from the maternal or paternal side.
(99) There is a real advantage for the technique herein described, in time of preparation of samples (no need to sow and wait for plantlets to analyze, no need to prepare DNA for a large number of samples) and cost.
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