CN113322331B - A method for assessing the potential applicability of insect-resistant rice varieties based on brown planthopper-specific virulence genotypes - Google Patents

Abstract

The invention discloses the relationship between the polymorphic site genotype of the brown planthopper cytochrome P450CYP4C61 gene and the nicotinic acetylcholine receptor α-7-like gene and the harm of the brown planthopper, and on this basis provides a specific harm of the brown planthopper based on the A method for assessing the potential applicability of insect-resistant rice varieties by sex genotype is more accurate and reliable than conventional assessment methods. The combination of genetic variation sites found in the present invention can divide any N. lugens field population into three genotypes related to virulence, and has extremely wide representation and applicability. Testing the insect resistance of target rice varieties can more objectively reflect the effectiveness of potential rice insect-resistant varieties when applied in the field in the future.

Description

A method for assessing the potential applicability of insect-resistant rice varieties based on brown planthopper-specific virulence genotypes

technical field

The invention belongs to the technical field of rice insect-resistant phenotype identification. More specifically, it relates to a method for assessing the potential applicability of an insect-resistant rice variety based on a specific virulence genotype of the brown planthopper.

Background technique

Brown planthopper is one of the main pests that harm rice, and its outbreak can lead to a large reduction in rice field production and seriously threaten food security. The selection and promotion of insect-resistant rice varieties is considered to be one of the important means to control brown planthopper damage and ensure food security, and has the advantages of economy, environmental protection and sustainability.

At present, in the identification process of rice insect-resistant phenotype, the genetic background of the pest itself is often not required. For example, Chinese patent CN 112083128A discloses a method for phenotypic identification of high-yield and pest-resistance of rice. It collects and analyzes hyperspectral images of rice treated with pests, and calculates the high-yield and pest-resistance of rice according to the formula, without making any requirements for pests . Chinese patent CN 111264320A discloses a method for accurate identification of brown planthopper resistance in the adult stage of rice. In the identification process, manual inoculation is required, but there are no special requirements for the inoculated pests. Most of the brown planthopper populations used in these methods are long-term laboratory-raised populations, which are often quite different from the actual genetic background of field populations. The bred varieties were quickly adapted to the field population after being applied in the field, and even did not have the proper resistance phenotype to the field population, which could not truly reflect the true resistance of the bred varieties to the field N. lugens population. level, resulting in the final use effect is not as expected. However, if the field population is simply used to conduct the rice insect resistance test, it is often difficult to obtain reliable and ideal results without knowing the genetic background of the pathogenicity of the field population. Therefore, in order to more accurately evaluate the insect resistance of rice varieties, it is necessary to clarify the genetic background related to the harmfulness of N. lugens populations in the field.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing above-mentioned technologies, and to provide a method for evaluating the potential applicability of insect-resistant rice varieties based on the specific harmful genotype of N. lugens.

The first object of the present invention is to provide the polymorphism site 88-254-352-542 of the brown planthopper cytochrome P450 CYP4C61 gene and the polymorphism site 576-580 of the nicotinic acetylcholine receptor α-7-like gene combined in Application in preparation of brown planthopper virulence genotype identification kit, identification of brown planthopper virulence level and assessment of potential applicability of insect-resistant rice varieties.

The second object of the present invention is to provide a kit for identifying the harmful genotype of N. lugens.

The third object of the present invention is to provide a method for identifying the harmful genotype of N. lugens.

A fourth object of the present invention is to provide a method for assessing the potential applicability of an insect-resistant rice variety based on a specific virulence genotype of N. lugens.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention first provides the polymorphism site 88-254-352-542 of the brown planthopper cytochrome P450 CYP4C61 gene shown in SEQ ID NO.5 and the nicotinic acetylcholine receptor α-7-like (nAChR) shown in SEQ ID NO.6 The relationship between the combination of polymorphism loci 576-580 of -α-7-like) gene and the virulence genotype of N. lugens is shown in the table:

Among them, Genotype1 is a non-toxic genotype, and Genotype2 and Genotype3 are harmful genotypes.

By detecting the polymorphism sites 261588-261754-261852-262042 of the brown planthopper cytochrome P450 CYP4C61 gene (ie, the 88th, 254th, 352nd and 542nd bases in the sequence of SEQ ID NO. 5) and nicotine The genotype of the polymorphism site 251576-251580 of the acetylcholine receptor α-7-like (nAChR-α-7-like) gene (that is, the 576th and 580th bases of the SEQ ID NO. 6 sequence) can be According to the table, judge whether the tested brown planthopper is a harmful genotype. Therefore, the present application protects the genotypes of the polymorphic sites 251576-251580 of the nicotinic acetylcholine receptor α-7-like gene of the brown planthopper and the polymorphic sites 261588-261754-261852-262042 of the cytochrome P450 CYP4C61 gene. Application of the brown planthopper virulence genotype, preparation of a brown planthopper virulence genotype identification kit, and/or evaluating the potential applicability of insect-resistant rice varieties.

The present invention provides an identification kit for the harmful genotype of N. lugens, which comprises detection of polymorphism sites 261588-261754-261852-262042 of N. lugens cytochrome P450 CYP4C61 gene (ie, the 88th position of the sequence of SEQ ID NO.5). , 254, 352 and 542 bases) and nicotinic acetylcholine receptor alpha-7-like gene polymorphism sites 251576-251580 genotype reagents.

The present invention also provides a method for identifying the harmful genotype of brown planthopper, comprising the following steps:

S1. Extracting the genomic DNA of brown planthopper;

S2. Take the DNA obtained in step S1 as a template, respectively carry out PCR amplification on the N. lugens nicotinic acetylcholine receptor α-7-like gene and the cytochrome P450 CYP4C61 gene fragment;

S3. After sequencing the amplified product, compare it with the above table.

Preferably, the sequences of PCR amplification primers used in step S2 are shown in SEQ ID NOs. 1 to 4, see Example 1.

Preferably, the PCR amplification reaction system used in step S2 is 1 μL of 20ng/μL template DNA, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 25 μL of 2×HiFi Mix, and 20 μL of ddH ₂ O, totaling 50 μL, see Example 1.

Preferably, the PCR amplification procedure described in step S2 is pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 40s for 30 cycles; and a final extension at 72°C for 5 minutes, see Examples 1.

The present invention further conducts rice insect resistance phenotype identification test by introducing brown planthopper populations with different virulence-related genotypes, thereby obtaining more real and reliable phenotype data, and can effectively evaluate the effectiveness level of rice insect resistance phenotype.

The present invention also provides a method for evaluating the potential applicability of an insect-resistant rice variety based on the specific harmful genotype of N. lugens, comprising the following steps:

S1. Take the N. lugens field population captured in the field as the F0 generation, and place it on the insect-sensitive rice variety to propagate for one generation to obtain the F1;

S2. After F1 grows to adults, pair the female and male adults in pairs, transfer them to the susceptible rice in the middle stage of tillering and raise them separately to ensure that the females and males mate and lay eggs for 7 days. Genomic DNA was extracted from a single head;

S3. Take the DNA obtained in step S2 as a template, respectively carry out PCR amplification of the brown planthopper cytochrome P450 CYP4C61 gene and the nicotinic acetylcholine receptor α-7-like gene;

S4. Sequence the obtained PCR product to obtain the genotypes of the corresponding SNPs on the individual CYP4C61 gene and nAChR-α-7-like gene of N. lugens;

S5. Compare the obtained genotypes with the above table, and obtain the offspring of the non-toxic genotypes Genotype1, the harmful genotypes Genotype2 and Genotype3, respectively, if the paired parental genotypes are of the same genotype and each If none of the points are heterozygous genotypes, their progeny can be retained as progeny of a specific homozygous genotype;

S6. Use the homozygous progeny of the three genotypes to conduct the insect resistance test on the target rice variety. If the target rice variety has resistance to the three genotypes of N. lugens, and the resistance level is higher than that of the homozygous progeny of different genotypes If there is no significant difference between the two genotypes, the target variety is considered to have relatively stable and excellent insect resistance; if the target rice variety has resistance to all three genotypes of BPH, but the resistance levels to the harmful genotypes Genotype2 and Genotype3 are significant. If the resistance level of the target rice variety is lower than the resistance level to the non-toxic genotype Genotype1 brown planthopper, it is considered that the target variety has insect resistance at present, but there is a risk of insect resistance failure; if the target rice variety is resistant to the non-toxic genotype Genotype1 brown planthopper The target variety is considered to have no field-applicable insect resistance if it has resistance, but does not have resistance to one or both of the brown planthoppers of the harmful genotypes Genotype2 and Genotype3.

Preferably, the sensitive rice variety is TN1, see Example 2.

The present invention also applies to protect the application of the kit and the method in assessing the potential applicability of insect-resistant rice varieties by N. lugens.

The present invention has the following beneficial effects:

The present invention discloses the genotypes of the polymorphic site 251576-251580 of the nicotinic acetylcholine receptor α-7-like gene of the brown planthopper and the polymorphic site 261588-261754-261852-262042 of the cytochrome P450 CYP4C61 gene and the damage caused by the brown planthopper Sex genotype relationship. On this basis, the homozygous progeny of the non-toxic genotypes Genotype 1 and the harmful genotypes Genotype 2 and 3 were obtained by screening the N. lugens caught in the field, and the target rice varieties were tested for insect resistance to evaluate the insect resistance. Potential suitability of rice varieties. Compared with the conventional evaluation method, this method overcomes the defect of not considering the actual genetic background of the field population of N. lugens in the conventional evaluation of the phenotype determination of rice insect-resistant varieties, and the results are more accurate and reliable. The combination of genetic variation sites found in the present invention can distinguish any N. lugens field population into three genotypes related to virulence, and has extremely wide representation and applicability. Using the homozygous progeny of the three genotypes of N. lugens populations to test the insect resistance of the target rice varieties can more objectively reflect the effectiveness of potential rice insect-resistant varieties in future field applications.

Description of drawings

Fig. 1 is a technical roadmap of the method for evaluating the potential applicability of insect-resistant rice varieties based on the specific harmful genotypes of N. lugens.

Figure 2 shows the resistance levels of different rice varieties to N. lugens determined by honeydew method.

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 Harmful genotype analysis of brown planthopper field populations in different regions

In order to verify the combination of genetic variation sites found in the present invention, any N. lugens field population can be divided into three genotypes related to virulence, which has extremely wide representation and applicability. In 2017, the inventors randomly sampled the field population of N. lugens in Shaoguan, Guangdong, Zunyi, Guizhou, and Zhoushan, Zhejiang, and analyzed the harmful genotypes of the collected N. lugens samples through the steps of extracting N. lugens DNA, PCR amplification and sequencing. Classification.

1. Genomic DNA extraction of brown planthopper

The present invention uses the Genomic DNAIsolation Kit of OMEGA company to extract genomic DNA, and the steps are as follows:

(1) Take the worms and grind them in liquid nitrogen thoroughly, and add 500 μL of Buffer GTC/2-Me;

(2) Vortex vigorously for 15s, let stand for 2-3min at room temperature, and the longest can be extended to 5min;

(3) Centrifuge at 13,000 rpm for 5 min;

DNA Column中，13000转离心1min；(4) Transfer the supernatant to

In the DNA Column, centrifuge at 13,000 rpm for 1 min;

DNA Column套在2mL收集管中，加500μL Buffer HB到柱子中，13000转离心1min，弃滤液；(5) put the

Put the DNA Column in a 2mL collection tube, add 500μL of Buffer HB to the column, centrifuge at 13,000 rpm for 1 min, and discard the filtrate;

(6) Add 700 μL of DNA Wash buffer to the column, centrifuge at 13,000 rpm for 1 min, and discard the filtrate;

(7) Put the column back into the collection tube, centrifuge at 12,000 rpm for 2 min, and dry the column;

(8) Transfer the column to a new 1.5 mL centrifuge tube, add 50-100 μL of Elution Buffer, place at room temperature for 2 min, and centrifuge at 13,000 rpm for 1 min.

2. PCR amplification of nAChR-α-7-like gene and CYP4C61 gene of N. lugens

Using the above-mentioned N. lugens genomic DNA as a template, the following primers were used to amplify the fragments containing the corresponding SNPs by PCR.

The primers were designed with the nicotinic acetylcholine receptor α-7-like (nAChR-α-7-like) gene (KN152279.1) and cytochrome P450 CYP4C61 gene (KN152684.1) of N. lugens as target genes, respectively.

The amplification primer sequence of the nAChR-α-7-like gene is:

N-F: CATTATTCATACAGATTTGATGGAGAG (SEQ ID NO. 1);

N-R: CACTAGGTGTCAAATCTACAAAACC (SEQ ID NO. 2).

The amplification primer sequences of the CYP4C61 gene are:

P-F: TCGAAGGTGAGTTGTTTGTTTTGT (SEQ ID NO. 3)

P-R: AGCCCTAAAGAGAAAAGAGACAATGT (SEQ ID NO. 4)

Use KAPA's HiFiHotStartReadyMix PCR Kit reagent for PCR amplification, and add the reagents shown in the following table to a 0.2mL PCR centrifuge tube:

PCR reaction program: heating the lid, pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 40s for 30 cycles; the final extension at 72°C for 5 min.

3. Individual genotyping of brown planthopper

The above PCR products were sent to a sequencing company for direct sequencing, and the sequences were analyzed according to the sequencing peak map.

Analysis of the combination of target SNP sites, including 261588, 261754, 261852 and 262042 of the CYP4C61 gene (corresponding to the 88th, 254th, 352nd and 542nd bases in the sequence of SEQ ID NO.5, respectively base). And two sites 251576 and 251580 of the nAChR-α-7-like gene (corresponding to the 576th and 580th bases in the sequence of SEQ ID NO. 6, respectively). The relationship between the polymorphism site 88-254-352-542 of N. lugens cytochrome P450 CYP4C61 gene and the polymorphic site 576-580 of nAChR-α-7-like gene and the harmful genotype of N. lugens are shown in Table 1 As shown, if the combination of target SNP sites conforms to the combination characteristics of Genotype1 in Table 1, the individual genotype of the brown planthopper is the non-toxic genotype Genotype1; if the combination of the target SNP sites conforms to the combination characteristics of Genotype2 in Table 1 , the individual genotype of the brown planthopper is the harmful genotype Genotype2; if the combination of the target SNP loci meets the combination characteristics of Genotype3 in Table 1, the individual genotype of the brown planthopper is the harmful genotype Genotype3.

Table 1 The relationship between polymorphism sites and the harmful genotypes of brown planthopper

The results of the genotype analysis of the field populations of N. lugens in different regions are shown in Table 2. There are a total of 263 samples, which can be classified into one of three genotypes related to virulence by using the combination of genetic variation sites found in the present invention. , indicating that the combination of genetic variation loci found in the present invention can distinguish any N. lugens field population into three genotypes related to virulence, and has extremely wide representation and applicability.

Table 2 Harmful genotype analysis of brown planthopper field populations in different regions

Genotype1 Genotype2 Genotype3 total Shaoguan, Guangdong 19 46 twenty one 86 Zunyi, Guizhou 27 54 9 90 Zhoushan, Zhejiang 26 52 9 87

Example 2 Method for assessing the potential applicability of insect-resistant rice varieties based on the specific virulence genotypes of the brown planthopper

A method for evaluating the potential applicability of insect-resistant rice varieties based on the specific virulence genotypes of the brown planthopper, the technical roadmap is shown in Figure 1.

1. Treatment of brown planthopper field population;

The N. lugens field populations P-SG were captured in the field of Shaoguan, Guangdong, respectively, and placed on the susceptible rice variety TN1 to propagate for one generation.

After the worms grow into adults, the female adults and male adults are paired in pairs, and transferred to TN1 rice in the middle stage of tillering to be reared separately. After ensuring that the female worms mate with the male worms and lay eggs for 7 days, the parental adult worms are removed for subsequent use. Genomic DNA extraction.

2. Single-headed N. lugens genome DNA extraction;

3. PCR amplification of nAChR-α-7-like gene and CYP4C61 gene of N. lugens;

4. Individual genotyping of brown planthopper;

The specific experimental steps 2-4 were carried out with reference to Example 1.

5. Obtaining the progeny of the brown planthopper homozygous genotype

If the paired parental genotypes are the same genotype and each site is not a heterozygous genotype, the progeny insects can be retained as the specific homozygous genotype progeny. Homozygous progeny of Genotype1, Genotype2 and Genotype3 genotypes.

6. Determination of resistance levels of different rice varieties to homozygous progeny of three genotypes

Taking the susceptible variety TN1 rice as control, the resistance levels of rice varieties IR36 and Rathu Heenati (R.H.) to different genotypes of N. lugens were determined by honeydew method.

The specific operation steps of honeydew detection are as follows: move a single plant of rice seedlings 30 days after sowing into a plastic cup (15cm × 10cm), remove the tillers and leave the main stem, and remove the brown planthopper adults that have been starved for 3 hours for one day. The winged females were inserted into the wax-film pouches with a straw, and each wax-film pouch was connected with one female worm, and then the wax-film pouches were wrapped on the rice plants of each treatment, and each treatment was repeated 5 times. The parafilm pouches were weighed with an electronic balance before inoculation. After 24 hours, the wax film sachet was removed and weighed again, and the difference between the two weights was the honeydew secretion of N. lugens. According to the amount of honeydew secreted by the brown planthopper after feeding on a specific rice variety, the resistance of the rice variety to the individual brown planthopper can be determined.

The evaluation results are shown in Figure 2. After feeding on the control variety TN1, the brown planthopper individual had the highest amount of honeydew secretion, and there was no significant difference between different genotypes. It also proved that the control variety TN1 had no resistance to the three genotypes of the brown planthopper. . The candidate insect-resistant variety IR36 has high resistance to the brown planthopper of Genotype1, but weak resistance to the brown planthopper of Genotype2, so it can be considered that it has no applicability in the field. The candidate insect-resistant variety R.H. has high resistance to all three genotypes of N. lugens, so it can be used as an effective insect-resistant variety, but the amount of honeydew fed on R.H. by Genotype2 was significantly higher than that of Genotype1, indicating that The R.H. variety has the risk of losing its resistance to insects in the future, and this result is consistent with the research results of Zhang Yang et al. J]. Guangdong Agricultural Science, 2011(21):28-30.).

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

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Claims (9)

1. The application of the reagent for detecting the combination of the polymorphic sites 88-254-352-542 of the brown planthopper cytochrome P450CYP4C61 gene shown in SEQ ID NO.5 and the polymorphic site 576-580 of the nAChR-alpha-7-like gene shown in SEQ ID NO.6 in identifying the harmful genotype of the brown planthopper or preparing an identification kit of the harmful genotype of the brown planthopper is characterized in that the relationship between the polymorphic site combination genotype and the harmful genotype of the brown planthopper is shown in the following list:

wherein Genotype1 is a non-deleterious Genotype, and Genotype2 and Genotype3 are deleterious genotypes.

2. A method for identifying the pest causing genotype of brown planthopper, which is characterized by comprising the following steps:

s1, extracting genome DNA of brown planthopper;

s2, respectively carrying out PCR amplification on the brown planthopper cytochrome P450CYP4C61 gene and the nicotinic acetylcholine receptor alpha-7-like gene by taking the DNA obtained in the step S1 as a template;

s3, after sequencing of the amplification products, comparing the amplification products with the table of claim 1; if the combination of the target SNP loci conforms to the combination characteristics of Genotype1 in the table, the individual Genotype of the brown planthopper is non-deleterious Genotype 1; if the combination of the target SNP loci meets the combination characteristics of Genotype2 in the table, the individual Genotype of the brown planthopper is a harmful Genotype 2; and if the combination of the target SNP loci meets the combination characteristics of Genotype3 in the table, the individual Genotype of the brown planthopper is the deleterious Genotype 3.

3. The method according to claim 2, wherein the PCR amplification primer sequence used in step S2 is shown in SEQ ID NO. 1-4.

4. The method of claim 3, wherein the PCR amplification reaction system used in step S2 is: 20 ng/. mu.L template DNA 1. mu.L, 10. mu.M forward primer 2. mu.L, 10. mu.M reverse primer 2. mu.L, 2 XHiFi Mix 25. mu.L, ddH₂O20. mu.L, 50. mu.L in total.

5. The method according to claim 3, wherein the PCR amplification procedure of step S2 is pre-denaturation at 95 ℃ for 2 min; 30 cycles of denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40 s; finally, the extension is carried out for 5min at 72 ℃.

6. The application of the method of any one of claims 2 to 5 in evaluating the potential applicability of insect-resistant rice varieties is characterized in that the potential applicability of the insect-resistant rice varieties is evaluated by capturing brown planthoppers from fields, identifying and screening homozygous progeny of non-lethal Genotype1 and lethal Genotype genotypes 2 and 3 by the method of any one of claims 2 to 5, and performing insect-resistant tests on target rice varieties.

7. A method for evaluating the potential applicability of an insect-resistant rice variety based on a specific injurious genotype of brown planthopper is characterized by comprising the following steps:

s1, taking field population of brown planthopper captured in the field as F0 generation, and placing the brown planthopper on pest-sensitive rice variety for propagation for one generation to obtain F1;

s2, after F1 grows to be an adult, pairing female adults and male adults pairwise, transferring the adult females and the male adults onto pest-sensitive rice for independent feeding, and taking away parent adults and respectively extracting genome DNA (deoxyribonucleic acid) in a single-head mode after the female and male adults are mated and lay eggs for 7 days;

s3, respectively carrying out PCR amplification on the alpha-7-like gene of the nicotine acetylcholine receptor of the brown planthopper and the cytochrome P450CYP4C61 gene fragment by taking the DNA obtained in the step S2 as a template;

s4, sequencing the obtained PCR product to obtain genotypes of corresponding SNPs on a CYP4C61 gene and a nAChR-alpha-7-like gene of the brown planthopper individual;

s5, comparing the obtained genotypes with the table of claim 1 to respectively obtain filial generations of a non-deleterious Genotype1, a deleterious Genotype2 and a Genotype3, wherein if the matched parental genotypes are of the same type and each locus is not of a heterozygous Genotype, the filial generations can be reserved as specific homozygous Genotype filial generations;

s6, respectively using homozygous offspring of the three genotypes to perform insect resistance tests on the target rice variety, and if the target rice variety has resistance to brown planthoppers of the three genotypes and the resistance level has no obvious difference among homozygous offspring of different genotypes, considering that the target rice variety has stable and excellent insect resistance; if the target rice variety has resistance to the brown planthoppers of three genotypes, but the resistance level to the brown planthoppers of the lethal genotypes Genotype2 and Genotype3 is obviously lower than the resistance level to the brown planthoppers of the non-lethal genotypes Genotype1, the target rice variety is considered to have insect resistance but has the risk of insect resistance failure at present; if the target rice variety has resistance to brown planthopper of non-deleterious Genotype1, but does not have resistance to one or both of brown planthopper of deleterious Genotype2 and Genotype3, the target rice variety is considered to have no insect resistance applicable in the field.

8. The method of claim 7, wherein the insect-susceptible rice variety is TN 1.

9. The method according to claim 7, wherein the transgenic rice susceptible to the tillering stage in step S2 is separately reared.

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