CN108796034B - Comparative hybrid population sampling method for establishing safety detection of transgenic drift environment - Google Patents
Comparative hybrid population sampling method for establishing safety detection of transgenic drift environment Download PDFInfo
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Abstract
The invention belongs to the technical field of transgenic organism environmental risk assessment. In particular to a comparative hybrid population sampling method for establishing the safety detection of a transgenic drifting environment. The method of the invention utilizes the transgene from the crop and a neutral molecular marker which is from the same chromosome of the crop, is close to the transgene and has no physical linkage to sample, respectively constructs a segregation experimental population of hybrid offspring which contains the transgene or does not contain the transgene, leads the genetic backgrounds of the two populations to be infinitely close, and then carries out analysis and comparison of relevant characters of fitness under the environmental condition of a homogeneous garden on the two populations, thereby accurately predicting and evaluating whether the transgene which enters the wild kindred species of the crop through natural hybridization can cause environmental risks. The method greatly reduces the difference of allele segregation between comparative groups (P + and P-) and the phenomenon of inconsistent genetic background caused by sampling, so that the result of the safety evaluation of the transgenic organisms is closer to the real situation.
Description
Technical Field
The invention belongs to the technical field of transgenic organism environmental risk evaluation and analysis, and particularly relates to a comparative hybrid population sampling method for establishing transgenic drift environment safety detection.
Background
The rapid development of transgenic technology continuously promotes the cultivation of transgenic plants, and the commercial planting of transgenic crops provides a new way for improving the crop yield, reducing the pesticide application, improving the agricultural ecological environment and solving the problem of food safety. However, transgenes may escape from the crop to its wild relative population through gene drift, which may present some environmental risk. Therefore, potential environmental risks that may be posed after a transgenic shift must be detected and evaluated before commercialization of the transgenic crop to ensure safe and sustainable use of the transgenic crop. The evaluation of the environmental risk possibly brought by the transgene drift of the transgenic crop is based on whether the fitness of the wild kindred population is changed after the transgene enters the wild kindred population of the crop through the gene drift, namely: by carrying out fitness-related trait analysis comparison on crop-wild relative hybrid progeny segregation experimental populations containing transgenes or not containing the transgenes under the environmental conditions of homogeneous gardens, whether the transgenes entering the wild relative of crops through natural hybridization (gene drift) can cause environmental risks or not is predicted and evaluated.
In the traditional method for sampling the hybrid progeny individuals for detecting the fitness effect of transgenic crops, individuals containing or not containing transgenes in a crop-wild allied hybrid segregation progeny population are identified by using a transgene as a marker, then the hybrid individuals containing or not containing the transgenes are respectively and randomly extracted from the segregation populations of the hybrid progeny, and a comparison population with the same number of individuals and containing or not containing the transgenes is established. However, this traditional sampling method results in differences in segregation from the parental alleles and genetic background inconsistencies between the two compared populations, namely: the transgenic-containing population possesses more crop alleles, while the population not containing the transgene possesses more crop wild-type sibling alleles. Besides the difference between the two populations containing the transgenes, the two populations also show inconsistency on genetic background, so that the result of fitness evaluation cannot truly and accurately reflect the fitness effect brought by the transgenes. Therefore, there is a need to establish a more reasonable population sampling method to ensure that the obtained comparison population (P +) containing the transgene and the comparison population (P-) not containing the transgene should be very close to each other in genetic background except for the difference in the presence or absence of the transgene, so that the fitness effect caused by the transgene can be truly evaluated, and whether the transgene entering the wild related species of the crop through gene drift would cause environmental risk or not can be accurately predicted.
Disclosure of Invention
The invention aims to provide a sampling method for establishing fitness comparison hybrid population for safety detection of a transgenic drift environment, so as to reduce the phenomena of partial separation of alleles from parents and inconsistent genetic background caused by sampling (manual selection) in the traditional sampling method for constructing a comparison population containing a transgene and a comparison population without the transgene, and enable the evaluation result to be closer to the real situation.
Whether a transgene wanders away presents an environmental risk depends in large part on whether the transgene, after wandering into the wild relative of the crop, results in a significant change in the fitness of the wild relative of the crop (which refers to the ability of an organism to survive and transmit its gene to progeny under certain conditions). While a greater increase in fitness (fitness benefit) or decrease in fitness (fitness cost) may result in different environmental risks, the accuracy of such risk detection depends on whether the genetic background is the same between the comparison populations (P + and P-). The invention establishes a novel method for sampling a comparative hybrid population for safely detecting a transgenic drift environment, and the method comprises the step of hybridizing crop-wild allied species containing transgenes and crop-wild allied species not containing the transgenesGeneration segregation experimental population (F)2，F3) The obtained comparison population (P +) containing the transgenes is ensured to be very close to the genetic background except for the difference in the aspect of containing the transgenes with the comparison population (P-) without the transgenes by sampling for 2 times, so that the purpose of truly evaluating the fitness effect of the transgenes is achieved, and the method is further used for accurately predicting and evaluating whether the transgenes entering the wild kindred species of the crops through natural hybridization (gene drift) can cause environmental risks.
The invention provides a comparative hybrid population sampling method for establishing safety detection of a transgenic drifting environment, which comprises the following specific steps:
(1) in the self-separation population of hybrid progeny of any transgenic crop and its wild relative species, the transgenic gene is used as molecular marker to sample the hybrid individuals containing the transgenic gene in the population, and a comparison population (namely a transgenic positive population P +) containing the transgenic gene is formed;
(2) in the same self-separating population of hybrid progeny, a neutral molecular marker from a crop parent and the transgene are used as molecular markers to sample the hybrid individuals, and a comparative population (namely a transgenic negative population P-) which does not contain the transgene and has the same number with the transgenic positive population is formed; the method and the process for establishing the P group are as follows:
A. selecting a neutral molecular marker which is from a crop parent and is located on the same chromosome with the transgene, is close to the neutral molecular marker but has no physical linkage, and sampling hybrid individuals containing the neutral molecular marker in a population to form a population (marker population Pm) containing the neutral molecular marker;
B. in the marker population Pm, the transgene is used as a molecular marker to identify hybrid individuals, hybrid individuals containing the transgene in the population are eliminated, the population does not contain the transgene any more, and thus a comparative population (transgenic negative population P-) which has the same number of individuals as the transgenic positive population P + and does not contain the transgene is formed;
(3) and (3) comparing the fitness related traits of the P + population and the P-population by using a homogeneous garden experiment, and predicting whether the transgene enters a crop wild kindred population through gene drift to cause environmental risk or not according to the fitness comparison result of the P + population and the P-population.
The method of the invention uses the transgene as the molecular marker, and simultaneously uses a neutral molecular marker which is located on the same chromosome with the transgene and is very close to the transgene but has no physical linkage to sample the experimental population of the separated offspring of the transgenic crop-wild relative hybrid for 2 times, and respectively forms a comparative population (P +) containing the transgene and a comparative population (P-) not containing the transgene. The novel sampling method for comparing the hybrid populations for detecting the fitness effect of the transgenic crops, provided by the invention, reduces the phenomena of partial separation difference of the parents alleles and inconsistent genetic background among the comparison populations caused by sampling in the safety evaluation process of the transgenic shifting environment, thereby avoiding or reducing the error of analysis of the transgenic fitness effect, enabling the evaluation result to be closer to the real situation, and having good application prospect in the aspects of gene shifting of the transgenic crops and environmental risk evaluation.
Drawings
FIG. 1 is a graph showing the genetic background difference between populations compared to each other by testing the fitness effect of transgenic positive (P +) and transgenic negative (P-) hybrid populations using conventional sampling methods for transgene fitness analysis. C is the genome of the crop and W is the genome of the wild species.
FIG. 2 is a graph showing the fitness effect of transgenic positive (P +) and transgenic negative (P-) hybrid populations tested using the sampling method of the present invention for transgene fitness analysis, with the genetic background between the P + and P-populations being very close. C is the genome of the crop and W is the genome of the wild species.
FIG. 3 (A) shows the use of a conventional method for sampling a population of hybrid species with a cowpea trypsin inhibitor insect-resistant transgene (A)CpTI) Sampling for molecular markers to form a sample comprisingCpTITransgenic (positive population, P +) and nullCpTIAllele frequencies were inconsistent between the two comparative experimental populations of transgenics (negative population, P-); FIG. 3(B) is a schematic view showing the use of the present inventionThe method of sampling a population of hybrid species of (1), the resulting product comprisingCpTIThe transgene (positive population, P +) was compared to a gene containing the neutral marker RM10289 but not containing itCpTIThe allele frequencies between the two comparative experimental populations of transgene (negative population, P-) are very close.
Detailed Description
Example 1
Insect-resistant transgenes containing the same constructed using conventional sampling methodsCpTI(Positive population, P +) and absence of insect-resistant transgeneCpTI(negative population, P-) comparing experimental population, in the process of genetic analysis of P + population and P-population, it is found that P + and P-comparing population show different frequency of alleles from parents on partial SSR sites, and have segregation phenomenon, and further, the genetic differentiation index between P + and P-comparing population is calculated (PF ST ) The values indicate that there is a significant difference in genetic background between the compared populations (FIG. 1), which can severely affect the transgene against the insectCpTIAnalysis of the fitness effect.
Construction of a transgene comprising an insect resistance Using conventional sampling methodsCpTI(Positive population, P +) and absence of insect-resistant transgeneCpTI(negative population, P-) by the following steps:
(1) determining the hybridization combination: insect-resistant transgenic rice (CC) × weedy rice (WW);
(2) obtaining hybrid F containing insect-resistant transgene by artificial hybridization1Population (CW);
(3) for hybrid F1Bagging and selfing the population to obtain F2Isolating the population;
(4) to F2Separating the population to perform molecular identification of the insect-resistant transgene to obtain positive F containing the insect-resistant transgene2Individuals (F)2(+) and negative F without transgene2Individuals (F)2-）；
(5) Respectively mix F2+ and F2-the same number of individual random components comprisesCpTITransgenic (positive population, P +) and nullCpTITransgene (negative population)P-).
From F2Randomly selecting 1000 individuals from the segregating population for germination and extracting DNA, and utilizingCpTIUsing transgenes as markers, designing corresponding transgene screening primers (CpTI 1), and carrying out Polymerase Chain Reaction (PCR) and agarose gel electrophoresis detection on the DNA, wherein all individuals with 415bp electrophoresis bands are identified as containingCpTITransgenic individuals (F)2(+) from which 200 individuals were randomly drawn (F)2(+) consist ofCpTIA positive (P +) comparison population of transgenes; from the rest of the composition containingCpTIRandomly selecting 200 individuals from transgenic individuals, wherein the composition does not compriseCpTINegative (P-) comparison population of transgenes.
The experimental materials are shown in table 1.
Table 1 experimental materials contained in example 1
Randomly selecting 40 pairs of SSR molecular markers with polymorphism in cultivated rice and weedy rice parents, respectively calculating allele frequencies of each SSR locus in a P + population and a P-population from cultivated rice parents (CC) and weedy rice parents (WW) through PCR and SSR genotyping, identifying loci with significant deviation from Mendelian segregation (i.e. partial segregation) by using chi square test, and calculating the average allele frequency of SSR loci which are partially segregated in the P + population and the P-population; on this basis, the genetic differentiation index between the P + population and the P-population was calculated: (F ST ) The genetic background difference of the two comparative populations was evaluated as a genetic differentiation index value. The results are as follows:
1. the P + population and the P-population produced segregation of alleles at 12 SSR loci, wherein the P + population exhibited allele bias towards the oryza sativa parent (CC), i.e., allele C had a higher frequency value than allele W; the P-population showed a bias in allele towards the weedy rice parent (WW), i.e., the frequency value of allele C was lower than the frequency value of allele W (Table 2).
TABLE 2 contains insect-resistant transgenes (CpTI) Comparison population of (P +) and comparison population without transgene (P-) Gene frequency values for oryza sativa allele C and weedy rice allele W at 12 SSR loci that are partially segregated (chi-square test)pAll values are less than 0.01)
2. The average frequency of alleles C from the oryza sativa parents in the P + population and the P-population were statistically calculated, and it was found that the average frequency of alleles C from oryza sativa in the P + population was higher than that in the P-population (FIG. 3A), and that allele bias occurred in both the P + population and the P-population.
3. Calculating the genetic differentiation index value between the P + group and the P-group according to the genotype data matrix of each SSR marker of the P + group and the P-groupF ST ：
Wherein the content of the first and second substances,H T is F2The expected heterozygosity when the experimental population is in hardtemperature equilibrium is separated,H S calculating the expected heterozygosity when the P + group and the P-group are in the temperature balanceF ST A value of 0.025 indicates that significant genetic differentiation occurred between the P + population and the P-population, i.e., there was a significant difference in the genetic background of the two populations.
4. Because the genetic backgrounds of the P + population and the P-population are obviously different, in the process of utilizing a homogeneous garden experiment to evaluate environmental biological safety, whether the difference of related characters of fitness caused by the genetic background difference of a comparative population or a transgenic population cannot be accurately evaluatedCpTICausing it to be. Therefore, the results obtained by using P + and P-comparative experimental groups constructed by the conventional sampling method and performing the fitness analysis method may be applied to the transgenesCpTIIs suitable forThe degree effect generates misjudgment and influences us to transgenesCpTIEnvironmental risk assessment of drifting to wild kindred species of crops.
Example 2
Construction of a transgene comprising an insect resistance Using the sampling method of the present inventionCpTI(Positive population, P +) and absence of insect-resistant transgeneCpTI(negative population, P-). During the genetic analysis of the P + population and the P-population, it was found that the P + and P-comparison populations were at some SSR sites, and although it appeared that alleles from parents had a segregation phenomenon, the genetic differentiation index between the P + population and the P-comparison population ((F ST ) The value is very small, which indicates that the genetic background between the P + population and the P-population is very close (FIG. 2), and the detected fitness difference can truly reflect the difference brought by the transgenes, thereby being beneficial to the pairingCpTICorrect assessment of environmental risk of drifting of insect-resistant transgenes into wild kindred species of crops.
Construction of a transgene comprising an insect resistance Using the sampling method of the present inventionCpTI(Positive population, P +) and lack of an anti-insect transgeneCpTIThe procedure for comparing the experimental populations (negative population, P-) was as follows:
(1) determining the hybridization combination: herbicide-resistant transgenic rice (CC) × weedy rice (WW);
(2) obtaining hybrid F containing insect-resistant transgene by artificial hybridization1Population (CW);
(3) for hybrid F1Bagging and selfing the population to obtain F2Isolating the population;
(4) to F2Separating the population to perform molecular identification of the insect-resistant transgene to obtain positive F containing the insect-resistant transgene2Individuals (F)2(+) and negative F containing no insect-resistant transgene2Individuals (F)2-）；
(5) Utilizing an SSR molecular marker (and insect-resistant transgene) from cultivated riceCpTIClosely spaced but not physically linked neutral molecular markers on the same chromosome) to F2Identifying individuals of segregating population to obtain SSR molecular markers of cultivated riceF2Segregating population (Pm) and F without cultivated rice SSR molecular marker2Segregating population (Pm 1);
(6) insect-resistant transgenesis for Pm populationCpTIObtaining an individual (Pm +) containing the insect-resistant transgene and an individual (Pm-) not containing the insect-resistant transgene;
(7) respectively mix F2+ and Pm-individuals with the same random composition number containing the insect-resistant transgeneCpTI(Positive population, P +) and absence of transgeneCpTI(negative population, P-).
The experimental materials are shown in table 3.
Table 3 experimental materials contained in example 2
From F2Randomly selecting 2000 individuals from the segregating population for germination and DNA extraction, and using insect-resistant transgenosisCpTIAs a screening marker, a corresponding transgenic screening primer (CpTI 1) is designed, the DNA is subjected to Polymerase Chain Reaction (PCR) and agarose gel electrophoresis detection, and all individuals with 415bp electrophoresis bands are identified as containingCpTITransgenic individuals (F)2(+) randomly extracting 200 individuals (F)2(+) consisting of an insect-resistant transgeneCpTIComparative population (P +).
From F2Randomly selecting 2000 individuals from the segregating population for germination and extracting DNA, and utilizing SSR molecular marker RM10289 (and transgene) of cultivated riceCpTIOn the same chromosome and at a distance from the physical location of the transgene<300kb, polymorphic in cultivated rice population and weedy rice population) and transgenic primer CpTI1, all the individuals with cultivated rice RM10289 electrophoresis band (88 bp) and no insect-resistant transgenic characteristic electrophoresis band (415 bp) are composed of individuals without insect-resistant transgenic geneCpTIF of (A)2Separating individual (Pm-), randomly drawing 200 individuals (Pm-), and forming transgene without insect resistanceCpTIComparison group (P-)。
Sequence information for SSR molecular marker (RM 10289) is as follows:
forward primer (5 '-3'): CTTGATTGGCTCTTCTGTCAATGG
Reverse primer (5 '-3'): GAATTCGATCTGCATCTGTCACG are provided.
Randomly selecting 40 pairs of SSR molecular markers with polymorphism in cultivated rice and weedy rice parents, respectively calculating allele frequencies of each SSR locus in a P + population and a P-population from cultivated rice parents (CC) and weedy rice parents (WW) through PCR and SSR genotyping, identifying loci with significant deviation from Mendelian segregation (i.e. partial segregation) by using chi square test, and calculating the average allele frequency of SSR loci which are partially segregated in the P + population and the P-population; on this basis, the genetic differentiation index between the P + population and the P-population was calculated: (F ST ) The genetic background difference of the two comparative populations was evaluated as a genetic differentiation index value. The results are as follows:
1. the P + and P-populations produced allelic bias at the 12 SSR sites, and the allelic bias for both populations appeared biased towards the cultivated rice parent (CC), i.e., the frequency value for allele C was higher than the frequency value for allele W (Table 4);
2. statistically calculating the mean frequencies of alleles from the oryza sativa parent (CC) in the P + and P-populations, and finding that there is little difference between the mean frequencies of alleles from the oryza sativa parent (CC) in the P + and P-populations (fig. 3B);
3. calculating a genetic differentiation index value between the P + group and the P-group according to the genotype data matrix of each SSR marker of the P + group and the P-group:
wherein the content of the first and second substances,H T is F2The expected heterozygosity when the experimental population is in hardtemperature equilibrium is separated,H S is the expected impurity when the P + population and the P-population are in Harvard equilibriumDegree of contact, calculateF ST Has a value of<0.0001, indicating that there is little genetic differentiation between the P + population and the P-population, i.e., the genetic backgrounds of these two populations are very close;
4. because the genetic backgrounds of the P + population and the P-population are very close, the detected difference of the traits related to fitness in the process of environmental biological safety evaluation in a homogeneous garden experiment is due to the transgeneCpTICausing it to be. Therefore, the fitness analysis result obtained by the sampling method of the invention truly reflects the fitness effect brought by transgenosis and avoids the transgenosisCpTIMisjudgment of fitness effect evaluation is favorable for the transgenic cellsCpTICorrect assessment of environmental risk of drifting to wild kindred species of crop.
TABLE 4 sampling with RM10289 molecular marker containing insect-resistant transgenesCpTIComparison population of (P +) and comparison population of non-insect-resistant transgene (P-) Gene frequency values (chi-square test) for oryza sativa allele C and weedy rice allele W at 12 SSR loci that are biased apartpAll values are less than 0.01)
Claims (1)
1. A comparative hybrid population sampling method for establishing safety detection of a transgenic drifting environment is characterized by comprising the following specific steps:
(1) in a hybrid progeny self-separation population of any transgenic crop and wild kindred species thereof, the transgene is used as a molecular marker, and a hybrid individual containing the transgene in the population is sampled to form a comparison population containing the transgene, namely a transgenic positive population P +;
(2) in the same self-separating population of hybrid progeny, a neutral molecular marker from a crop parent and the transgene are used as molecular markers to sample the hybrid individuals, and a comparative population which does not contain the transgene and has the same number with the transgenic positive population, namely a transgenic negative population P < - >, is formed; the method and process for establishing the P-population are as follows:
A. selecting a neutral molecular marker which is from a crop parent and is positioned on the same chromosome with the transgene, is close to the chromosome and has no physical linkage, and sampling hybrid individuals containing the neutral marker in a population to form a population containing the neutral marker, namely a marker population Pm;
B. in the marker population Pm, the transgene is used as a molecular marker to identify hybrid individuals, hybrid individuals containing the transgene in the population are eliminated, the transgene is not contained in the population, and thus a comparative population which does not contain the transgene and has the same number of individuals as the number of the individuals of the transgene positive population P + is formed, namely a transgene negative population P-;
(3) and (3) comparing the fitness related traits of the P + population and the P-population by using a homogeneous garden experiment, and predicting whether the transgene enters a crop wild kindred population through gene drift to cause environmental risk or not according to the fitness comparison result of the P + population and the P-population.
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