SNP (Single Nucleotide Polymorphism) sites for eight national traceability of bactrocera dorsalis as well as detection primer combination and application of SNP sites

Abstract

The invention discloses a group of SNP (Single Nucleotide Polymorphism) loci for eight national traceability of bactrocera dorsalis as well as a detection primer combination and application of the SNP loci. The technical problem to be solved is to perform national geographical tracing on bactrocera dorsalis. Specifically, the invention discloses any one of the following applications of the substance for detecting the genotype of SNP1-SNP8: A1) identifying or assisting in identifying the national geographical source of the tephritidae animal or preparing the substance for identifying or assisting in identifying the national geographical source of the tephritidae animal; and A2) identifying or assisting in identifying the national geographical population attribution of the bactrocera or preparing substances for identifying or assisting in identifying the national geographical population attribution of the bactrocera, and detecting the genotypes of the eight SNPs one by one to obtain the national geographical traceability of the bactrocera dorsalis.

Description

Technical Field

[0001] The present invention specifically relates to a group of SNP sites for tracing the origin of the citrus fruit fly in eight countries and a combination and application of detection primers thereof. Background Art

[0002] Single nucleotide polymorphism (SNP) refers to the variation of a single nucleotide in the genome. It is the smallest unit of variation and is a form of variation formed by the substitution, transversion, insertion or deletion of a single nucleotide pair.

[0003] The citrus fruit fly, Bactroceradorsalis (Hendel), belongs to the class Insecta, order Diptera, family Tephritidae, genus Bactrocera. It is one of the world's most important agricultural pests, attracting significant global attention and designated in my country as an import quarantine pest and a key agricultural invasive species under national control. It is a polyphagous fruit fly with the widest host range among the Bactrocera genus, infesting over 250 fruit and vegetable species, posing a serious threat to fruit and vegetable production.

[0004] The fruit fly (Bactrocera dorsalis) is native to tropical and subtropical Asia. It was first discovered in India orientalis and has since spread to 124 regions in 75 countries worldwide, including Asia, Africa, and North America. In China, the fruit fly was first discovered in Taiwan in 1911. Its robust egg-laying capacity, short life cycle, long-range flight (up to 50–100 km), wide host range, strong environmental adaptability, and competitive ability make it a highly invasive pest. Against the backdrop of global change and economic integration, the spread of the fruit fly is becoming increasingly serious. The pest has been repeatedly introduced into areas outside its natural range, attracting significant attention in the international fields of plant quarantine and invasion biology. However, it is often difficult to determine the source and transmission routes of fruit flies intercepted by port quarantine authorities and monitored in non-infested areas, complicating the development of invasion prevention and control strategies. Currently, a comprehensive set of single nucleotide polymorphisms (SNPs) and primer combinations for detecting the fruit fly at the national level are lacking. Therefore, it is necessary to develop a new technology to quickly and accurately analyze and trace the genetic diversity of fruit flies and clarify the sources of intercepted and monitored samples. Summary of the Invention

[0005] The technical problem solved by the present invention is to trace the origin of the citrus fruit fly.

[0006] In order to solve the above problems, the present invention provides the following applications.

[0007] Application, wherein the application is any of the following:

[0008] 1) Use of a substance for detecting the SNP1 genotype, a substance for detecting the SNP2 genotype, a substance for detecting the SNP3 genotype, a substance for detecting the SNP4 genotype, a substance for detecting the SNP5 genotype, a substance for detecting the SNP6 genotype, a substance for detecting the SNP7 genotype, and a substance for detecting the SNP8 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from Thailand;

[0009] 2) Use of substances for detecting the SNP1 genotype, substances for detecting the SNP2 genotype, substances for detecting the SNP3 genotype, substances for detecting the SNP4 genotype, substances for detecting the SNP5 genotype, substances for detecting the SNP6 genotype, and substances for detecting the SNP7 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from China;

[0010] 3) Use of a substance for detecting the SNP1 genotype, a substance for detecting the SNP2 genotype, a substance for detecting the SNP3 genotype, a substance for detecting the SNP4 genotype, a substance for detecting the SNP5 genotype, and a substance for detecting the SNP6 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from India;

[0011] 4) Use of a substance for detecting the SNP1 genotype, a substance for detecting the SNP2 genotype, a substance for detecting the SNP3 genotype, a substance for detecting the SNP4 genotype, and a substance for detecting the SNP5 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from Malaysia;

[0012] 5) Use of substances for detecting the SNP1 genotype, substances for detecting the SNP2 genotype, substances for detecting the SNP3 genotype, and substances for detecting the SNP4 genotype in identifying or assisting in identifying whether a product containing fruit flies to be tested originates from Vietnam;

[0013] 6) Use of substances for detecting the SNP1 genotype, substances for detecting the SNP2 genotype, and substances for detecting the SNP3 genotype in identifying or assisting in identifying whether a fruit fly species to be tested is a product originating from South Africa;

[0014] 7) Use of substances for detecting the SNP1 genotype and substances for detecting the SNP2 genotype in identifying or assisting in identifying whether a product containing fruit flies originates from the Philippines;

[0015] 8) Use of substances for detecting the SNP1 genotype in identifying or assisting in identifying whether a fruit fly species being tested is a product originating from the United States;

[0016] The SNP1 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 1 and is A or C;

[0017] The SNP2 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 2 and is A or T;

[0018] The SNP3 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 3 and is G or A;

[0019] The SNP4 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 4 and is A or G;

[0020] The SNP5 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 5, and is T or A;

[0021] The SNP6 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 6, and is A or T;

[0022] The SNP7 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 7, which is T or C;

[0023] The SNP8 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 8 and is T or A.

[0024] The genotype of SNP1 is CC, which indicates a homozygous C at position 300 of Sequence 1. The genotype of SNP2 is TT, which indicates a homozygous T at position 300 of Sequence 2. The genotype of SNP3 is AA, which indicates a homozygous A at position 300 of Sequence 3. The genotype of SNP4 is GG, which indicates a homozygous G at position 300 of Sequence 4. The genotype of SNP5 is AA, which indicates a homozygous A at position 300 of Sequence 5. The genotype of SNP6 is TT, which indicates a homozygous T at position 300 of Sequence 6. The genotype of SNP7 is CC, which indicates a homozygous C at position 300 of Sequence 7. The genotype of SNP8 is AA, which indicates a homozygous A at position 300 of Sequence 8.

[0025] The genotype of SNP1 is AA, which indicates the homozygous type for A at position 300 of Sequence 1. The genotype of SNP2 is AA, which indicates the homozygous type for A at position 300 of Sequence 2. The genotype of SNP3 is GG, which indicates the homozygous type for G at position 300 of Sequence 3. The genotype of SNP4 is AA, which indicates the homozygous type for A at position 300 of Sequence 4. The genotype of SNP5 is TT, which indicates the homozygous type for T at position 300 of Sequence 5. The genotype of SNP6 is AA, which indicates the homozygous type for A at position 300 of Sequence 6. The genotype of SNP7 is TT, which indicates the homozygous type for T at position 300 of Sequence 7. The genotype of SNP8 is TT, which indicates the homozygous type for T at position 300 of Sequence 8.

[0026] The genotype of SNP1 is AC, which is a heterozygous type with A at position 300 of sequence 1 and C at position 300 of sequence 1. The genotype of SNP2 is AT, which is a heterozygous type with A at position 300 of sequence 2 and T at position 300 of sequence 2. The genotype of SNP3 is GA, which is a heterozygous type with G at position 300 of sequence 3 and A at position 300 of sequence 3. The genotype of SNP4 is AG, which is a heterozygous type with A at position 300 of sequence 4 and G at position 300 of sequence 4. The genotype of SNP5 is TA, which is a heterozygous type with T at position 300 of sequence 5 and A at position 300 of sequence 5. The genotype of SNP6 is AT, which is a heterozygous type with A at position 300 of sequence 6 and T at position 300 of sequence 6. The genotype of SNP7 is TC, which is a heterozygous type with T at position 300 of sequence 7 and C at position 300 of sequence 7. The genotype of SNP8 is TA, which is a heterozygous type in which the 300th position of sequence 8 is T and the 300th position of sequence 8 is A.

[0027] In the above application, the fruit fly to be tested is Bactrocera dorsalis.

[0028] In the above, the substance may be a product. The detection substance may include reagents, kits and instruments for detecting the above-mentioned single nucleotide polymorphisms. Specifically, the primers and other reagents and instruments required for in vitro amplification of nucleic acids for detecting the above-mentioned single nucleotide polymorphisms.

[0029] In the above, Sequence 1 to Sequence 8 are genomic sequences. In the actual detection process, the genotypes of SNP1 to SNP8 can be detected and analyzed by detecting that Sequence 1 to Sequence 8 are genomic transcribed mRNA, mRNA reverse transcribed cDNA nucleotide sequence or the amino acid sequence of the encoded protein.

[0030] In order to solve the above problem, the present invention also provides a method.

[0031] The method is any of the following:

[0032] M1) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from Thailand, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from Thailand based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, SNP5 genotype, SNP6 genotype, SNP7 genotype, and SNP8 genotype as described above;

[0033] If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, the SNP7 genotype is not TT, and the SNP8 genotype is AA, the fruit fly to be tested is from Thailand or the candidate is from Thailand; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, the SNP7 genotype is not TT, and the SNP8 genotype is not AA, the fruit fly to be tested is not from Thailand or the candidate is not from Thailand;

[0034] M2) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from China, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from China based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, SNP5 genotype, SNP6 genotype, and SNP7 genotype as described above;

[0035] If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, and the SNP7 genotype is TT, the fruit fly to be tested is from China or the candidate is from China; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, and the SNP7 genotype is not TT, the fruit fly to be tested is not from China or the candidate is not from China;

[0036] M3) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from India, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from India based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, SNP5 genotype, and SNP6 genotype as described above;

[0037] If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, and the SNP6 genotype is TT, the fruit fly to be tested is from India or the candidate is from India; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, and the SNP6 genotype is not TT, the fruit fly to be tested is not from India or the candidate is not from India;

[0038] M4) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from Malaysia, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from Malaysia based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, and SNP5 genotype as described above;

[0039] If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, and the SNP5 genotype is AA, the fruit fly to be tested is from Malaysia or the candidate is from Malaysia; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, and the SNP5 genotype is not AA, the fruit fly to be tested is not from Malaysia or the candidate is not from Malaysia;

[0040] M5) a method for identifying or assisting in identifying whether a test fruit fly originates from Vietnam, comprising detecting a SNP genotype of the test fruit fly, and determining whether the test fruit fly originates from Vietnam based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, and SNP4 genotype as described above;

[0041] If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, and the SNP4 genotype is GG, the fruit fly to be tested is from Vietnam or the candidate is from Vietnam; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, and the SNP4 genotype is not GG, the fruit fly to be tested is not from Vietnam or the candidate is not from Vietnam;

[0042] M6) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from South Africa, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from South Africa based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, the SNP2 genotype, and the SNP3 genotype as described above;

[0043] If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, and the SNP3 genotype is AA, the fruit fly to be tested is from South Africa or the candidate is from South Africa; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, and the SNP3 genotype is not AA, the fruit fly to be tested is not from South Africa or the candidate is not from South Africa;

[0044] M7) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from the Philippines, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from the Philippines based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype and the SNP2 genotype as described above;

[0045] If the SNP1 genotype of the fruit fly to be tested is not CC and the SNP2 genotype is TT, the fruit fly to be tested is from the Philippines or the candidate is from the Philippines; if the SNP1 genotype of the fruit fly to be tested is not CC and the SNP2 genotype is not TT, the fruit fly to be tested is not from the Philippines or the candidate is not from the Philippines;

[0046] M8) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from the United States, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from the United States based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype described above;

[0047] If the SNP1 genotype of the fruit fly to be tested is CC, the fruit fly to be tested is from the United States or the candidate is from the United States; if the SNP1 genotype of the fruit fly to be tested is not CC, the fruit fly to be tested is not from the United States or the candidate is not from the United States;

[0048] The genotype of SNP1 is CC, which is a homozygous type with C at position 300 of sequence 1;

[0049] The genotype of SNP2 is TT, which is a homozygous type with T at position 300 of sequence 2;

[0050] The genotype of SNP3 is AA, which is a homozygous type with A at position 300 of sequence 3;

[0051] The genotype of SNP4 is GG, which is a homozygous type with G at position 300 of sequence 4;

[0052] The genotype of SNP5 is AA, which is a homozygous type with A at position 300 of sequence 5;

[0053] The genotype of SNP6 is TT, which is a homozygous type with T at position 300 of sequence 6;

[0054] The genotype of SNP7 is TT, which is a homozygous type with T at position 300 of sequence 7;

[0055] The genotype of SNP8 is AA, which is a homozygous type in which the 300th position of sequence 8 is A.

[0056] In the above, the United States may refer to Hawaii, USA. Malaysia may refer to Kuala Lumpur, Malaysia. The Philippines may refer to Dumaguete or Davao, Philippines. South Africa may refer to Mpumalanga, South Africa. Thailand may refer to Bangkok or Phuket. Vietnam may refer to Tien Giang, Vietnam. China may refer to Guangzhou, Guangdong, China; Wuhan, Hubei, China; Pingtung County, Taiwan, China; or Yilan County, Taiwan, China. India may refer to Assam, Bihar, Punjab, Telangana, or Tamil Nadu, India.

[0057] The SNP1 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 1 and is A or C;

[0058] The SNP2 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 2 and is A or T;

[0059] The SNP3 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 3 and is G or A;

[0060] The SNP4 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 4 and is A or G;

[0061] The SNP5 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 5, and is T or A;

[0062] The SNP6 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 6, and is A or T;

[0063] The SNP7 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 7, which is T or C;

[0064] The SNP8 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 8 and is T or A.

[0065] In M1) above, if the fruit flies to be tested do not originate from Thailand, then the fruit flies to be tested do not originate from the United States, the Philippines, South Africa, Vietnam, Malaysia, India and China.

[0066] In M2), if the fruit flies to be tested do not originate from China, then the fruit flies to be tested do not originate from the United States, the Philippines, South Africa, Vietnam, Malaysia and India.

[0067] In M3), if the fruit flies to be tested do not originate from India, then the fruit flies to be tested do not originate from the United States, the Philippines, South Africa, Vietnam and Malaysia.

[0068] In M4), if the fruit flies to be tested do not originate from Malaysia, then the fruit flies to be tested do not originate from the United States, the Philippines, South Africa and Vietnam.

[0069] In M5), if the fruit flies to be tested do not originate from Vietnam, then the fruit flies to be tested do not originate from the United States, the Philippines, and South Africa.

[0070] In M6), if the fruit flies to be tested do not originate from South Africa, then the fruit flies to be tested do not originate from the United States or the Philippines.

[0071] In M7), if the fruit flies to be tested do not originate from the Philippines, then the fruit flies to be tested do not originate from the United States.

[0072] Application of the above method in identifying or assisting in identifying the national geographical origin of Tephritidae animals.

[0073] In order to solve the above problems, the present invention also provides a product.

[0074] The product is any of the following:

[0075] P1) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from Thailand, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 as recited in claim 1;

[0076] P2) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from China, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7 as recited in claim 1;

[0077] P3) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from India, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, SNP5, and SNP6 as recited in claim 1;

[0078] P4) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from Malaysia, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, and SNP5 as recited in claim 1;

[0079] P5) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from Vietnam, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, and SNP4 as recited in claim 1;

[0080] P6) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from South Africa, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, and SNP3 as described in claim 1;

[0081] P7) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from the Philippines, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1 and SNP2 as described in claim 1;

[0082] P8) Identifying or assisting in identifying whether the fruit fly to be tested is a product originating from the United States, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is the SNP1 described in claim 1.

[0083] In the above-mentioned application, method, application or product, the product for detecting the genotypes of the eight SNPs SNP1-SNP8 is the following D1), D2), D3) or D4):

[0084] D1) containing in vitro nucleic acid amplification primers for specifically amplifying and detecting the SNP;

[0085] D2) an in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in D1);

[0086] D3) a kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2);

[0087] D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2) or the kit described in D3).

[0088] The in vitro nucleic acid amplification technology can be polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR) and nucleic acid sequence-dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent isothermal amplification technology (HDA) or Qβ replication technology.

[0089] The present application uses polymerase chain reaction (PCR) as an amplification method to perform polymorphism detection.

[0090] The specific amplification in D1) can detect the nucleotide sequence of the polymorphic sites SNP1-SNP8 by the presence or absence of amplification products or by the presence or absence of amplification products combined with auxiliary reagents such as probes.

[0091] In the above-mentioned applications, methods and products, the in vitro nucleic acid amplification primer may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Markers include but are not limited to dyes; radioactive labels, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence comprising the marker is detectable. In some embodiments, the nucleic acid is directly detected without a marker.

[0092] In the above-mentioned application, product or method, the in vitro nucleic acid amplification primers include a primer pair SNP1-F / SNP1-R for amplifying SNP1, a primer pair SNP2-F / SNP2-R for amplifying SNP2, a primer pair SNP3-F / SNP3-R for amplifying SNP3, a primer pair SNP4-F / SNP4-R for amplifying SNP4, a primer pair SNP5-F / SNP5-R for amplifying SNP5, a primer pair SNP6-F / SNP6-R for amplifying SNP6, a primer pair SNP7-F / SNP7-R for amplifying SNP7, and a primer pair SNP8-F / SNP8-R for amplifying SNP8;

[0093] The SNP1-F sequence is shown in Sequence 9; the SNP1-R sequence is shown in Sequence 10; the SNP2-F sequence is shown in Sequence 11; the SNP2-R sequence is shown in Sequence 12; the SNP3-F sequence is shown in Sequence 13; the SNP3-R sequence is shown in Sequence 14; the SNP4-F sequence is shown in Sequence 15; the SNP4-R sequence is shown in Sequence 16; the SNP5-F sequence is shown in Sequence 17; the SNP5-R sequence is shown in Sequence 18; the SNP6-F sequence is shown in Sequence 19; the SNP6-R sequence is shown in Sequence 20; the SNP7-F sequence is shown in Sequence 21; the SNP7-R sequence is shown in Sequence 22; the SNP8-F sequence is shown in Sequence 23; and the SNP8-R sequence is shown in Sequence 24.

[0094] In order to solve the above problems, the present invention also provides a DNA barcode of Bactrocera dorsalis.

[0095] The DNA barcodes are the 8 SNPs described in claim 1.

[0096] Use of the above-mentioned DNA barcode in identifying or assisting in identifying the national geographical origin of Tephritidae animals, or in preparing a material for identifying or assisting in identifying the national geographical origin of Tephritidae animals.

[0097] In order to solve the above problems, the present invention also provides a composition.

[0098] The composition contains one or more of a substance for detecting SNP1 genotype, a substance for detecting SNP2 genotype, a substance for detecting SNP3 genotype, a substance for detecting SNP4 genotype, a substance for detecting SNP5 genotype, a substance for detecting SNP6 genotype, a substance for detecting SNP7 genotype and a substance for detecting SNP8 genotype.

[0099] The fruit fly to be tested may be Bactrocera dorsalis. The Tephritidae animal may be Bactrocera dorsalis.

[0100] In the above, the oriental fruit fly may be from Kuala Lumpur, Malaysia, Mpumalanga, South Africa, Hawaii, USA, Davao, Philippines, Dumaguete, Philippines, Bangkok, Thailand, Phuket, Thailand, Tien Giang, Vietnam, Guangzhou, Guangdong, China, Zhangzhou, Fujian, China, Wuhan, Hubei, China, Pingtung County, Taiwan, China, Yilan County, Taiwan, China, Yuanjiang, Yunnan, China, Telangana, India, Assam, India, Bihar, India, Punjab, India, Tamil Nadu, India, Murree, Pakistan, Port Moresby, Papua New Guinea or Vientiane, Laos.

[0101] In the above, the United States may refer to Hawaii, United States. Malaysia may refer to Kuala Lumpur, Malaysia. The Philippines may refer to Dumaguete, Philippines or Davao, Philippines. South Africa may refer to Mpumalanga, South Africa. Thailand may refer to Bangkok, Thailand or Phuket, Thailand. Vietnam may refer to Tien Giang, Vietnam. China may refer to Guangzhou, Guangdong, China; Zhangzhou, Fujian, China; Wuhan, Hubei, China; Pingtung County, Taiwan, China; or Yilan County, Taiwan, China. India may refer to Assam, India; Bihar, India; Punjab, India; Telangana Pradesh, India; or Tamil Nadu, India. Pakistan may refer to Murree, Pakistan. Papua New Guinea may refer to Port Moresby, Papua New Guinea. Laos may refer to Vientiane, Laos.

[0102] Beneficial effects

[0103] Research on traceability technology contributes to a deeper understanding of the population structure, origin, and evolution of pests, providing crucial information and scientific foundations for pest quarantine identification, epidemic surveillance, integrated pest control, and trade dispute resolution. In microbial traceability research, traditional phenotyping techniques rely on culture, biochemical testing, and immunoassays. While simple to perform, these methods also have limitations, such as a limited number of phenotypic traits, high costs, and labor intensity. In recent years, the emergence of molecular markers based on DNA polymorphisms has provided effective detection tools for traceability research. Among these, third-generation molecular markers—SNPs—are currently the most effective methods for traceability. In particular, SNP data can be used to track the sources of invasion across geographical regions and determine the spread pathways of invasive species, providing a scientific basis for their prevention, control, and management. SNP markers are characterized by their wide distribution, strong representativeness, good genetic stability, and ease of automated analysis. With the increasing attention paid to SNP markers and the rapid development of molecular biology techniques, a number of rapid and efficient SNP typing methods have emerged.

[0104] The present invention discloses a set of SNP sites and detection primer combinations and applications for 8 national origins of the citrus fruit fly. The technical problem solved is to conduct national geographical tracing of the citrus fruit fly. Specifically disclosed is the application of a substance for detecting the genotypes of SNP1-SNP8 in any of the following: A1) identifying or assisting in identifying the national geographical origin of a Tephritidae animal or preparing a substance for identifying or assisting in identifying the national geographical origin of a Tephritidae animal; A2) identifying or assisting in identifying the national geographical population affiliation of a Tephritidae animal or preparing a substance for identifying or assisting in identifying the national geographical population affiliation of a Tephritidae animal. By detecting the genotypes of the above 8 SNPs one by one, the national geographical tracing of the citrus fruit fly can be obtained.

[0105] The present invention discovered eight SNP sites associated with the geographical origin of the citrus fruit fly, namely SNPs 1 to 8. SNP1 is the 300th nucleotide of sequence 1, which is A or C; SNP2 is the 300th nucleotide of sequence 2, which is A or T; SNP3 is the 300th nucleotide of sequence 3, which is G or A; SNP4 is the 300th nucleotide of sequence 4, which is A or G; SNP5 is the 300th nucleotide of sequence 5, which is T or A; SNP6 is the 300th nucleotide of sequence 6, which is A or T; SNP7 is the 300th nucleotide of sequence 7, which is T or C; and SNP8 is the 300th nucleotide of sequence 8, which is T or A.

[0106] The geographical origin of Bactrocera dorsalis was determined by the following method:

[0107] Detecting the genotypes of SNP1 to SNP8;

[0108] If the genotype of SNP1 is CC, the sample is from the United States;

[0109] If the genotype of SNP1 is not CC, continue to determine the genotype of SNP2. If the genotype of SNP2 is TT, the sample is from the Philippines.

[0110] If the genotype of SNP2 is not TT, continue to determine the genotype of SNP3. If the genotype of SNP3 is AA, the sample is from South Africa.

[0111] If the genotype of SNP3 is not AA, continue to determine the genotype of SNP4. If the genotype of SNP4 is GG, the sample is from Vietnam.

[0112] If the genotype of SNP4 is not GG, continue to determine the genotype of SNP5. If the genotype of SNP5 is AA, the sample is from Malaysia.

[0113] If the genotype of SNP5 is not AA, continue to determine the genotype of SNP6. If the genotype of SNP6 is TT, the sample is from India;

[0114] If the genotype of SNP6 is not TT, continue to determine the genotype of SNP7. If the genotype of SNP7 is TT, the sample is from China;

[0115] If the genotype of SNP7 is not TT, continue to determine the genotype of SNP8. If the genotype of SNP8 is AA, the sample is from Thailand;

[0116] The genotype of SNP1 is CC, which is a homozygous type with C at position 300 of sequence 1;

[0117] The genotype of SNP2 is TT, which is a homozygous type with T at position 300 of sequence 1;

[0118] The genotype of SNP3 is AA, which is a homozygous type with A at position 300 of sequence 1;

[0119] The genotype of SNP4 is GG, which is a homozygous type with G at position 300 of sequence 1;

[0120] The genotype of SNP5 is AA, which is a homozygous type with A at position 300 of sequence 1;

[0121] The genotype of SNP6 is TT, which is a homozygous type with T at position 300 of sequence 1;

[0122] The genotype of SNP7 is TT, which is a homozygous type with T at position 300 of sequence 1;

[0123] The genotype of SNP8 is AA, which is a homozygous type in which the 300th position of sequence 1 is A.

[0124] The aforementioned United States may refer to Hawaii, United States. The aforementioned Malaysia may refer to Kuala Lumpur, Malaysia. The aforementioned Philippines may refer to Dumaguete or Davao, Philippines. The aforementioned South Africa may refer to Mpumalanga, South Africa. The aforementioned Thailand may refer to Bangkok or Phuket. The aforementioned Vietnam may refer to Tien Giang, Vietnam. The aforementioned China may refer to Guangzhou, Guangdong, China; Zhangzhou, Fujian, China; Wuhan, Hubei, China; Pingtung County, Taiwan, China; or Yilan County, Taiwan, China. The aforementioned India may refer to Assam, Bihar, Punjab, Telangana, or Tamil Nadu, India. DETAILED DESCRIPTION

[0125] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0126] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0127] Example 1. Sample Collection, Genomic DNA Extraction and Next-Generation Sequencing Method

[0128] 1. Sample collection

[0129] All fruit fly specimens used in this study were collected from 18 geographic populations across eight countries: Malaysia, the Philippines, South Africa, Thailand, Vietnam, China, the United States, and India. Detailed sample information is provided in Table 1. All fruit fly specimens were collected from the field using the sex attractant methyl eugenol. After collection, the specimens were directly immersed in anhydrous ethanol and stored in an ultra-low-temperature freezer for long-term storage.

[0130] Table 18 countries' information on fruit fly samples

[0131]

[0132]

[0133] 2. Extraction of genomic DNA

[0134] Promega's Wizard SV Genomic DNA Purification System kit was used to extract genomic DNA from Bactrocera dorsalis. DNA quality and concentration were measured using an ultra-micro-UV spectrophotometer to ensure high-quality genomic DNA for next-generation sequencing library construction and sequencing.

[0135] 3. Next-generation sequencing methods

[0136] DNA samples that meet library construction requirements are sent to Beijing Berry Genomics Co., Ltd. for subsequent DNA quality testing. Once qualified, DNA is purified, mechanically sheared using a Bioruptor, end-repaired, and 3'-end PCR amplification is performed to construct a next-generation sequencing library with an average insert size of 450 bp. Paired-end sequencing with PE150 is performed using the Illumina Novaseq platform, ensuring a total of 6 Gb of raw data per fruit fly sample.

[0137] 2. SNPs detection and tracing SNP site screening in Bactrocera dorsalis resequencing data

[0138] 1. Quality control and filtering of Bactrocera dorsalis resequencing data

[0139] After obtaining the next-generation sequencing data, we used Fastp v0.20.0 to perform a quality check on the raw sequencing data, removing adapter sequences and low-quality sequences (such as those that were too short, had low base quality, or contained a large number of Ns). Default parameters were used.

[0140] 2. SNPs detection in Bactrocera dorsalis resequencing data

[0141] The present invention uses the genome of the citrus fruit fly (GCA_030710565.1) as a reference genome to detect SNPs. First, samtools is used to construct a reference sequence index, BWA is used to construct a reference sequence index file, and Picard is used to construct a reference sequence dictionary file. Then, the mem subroutine in BWA is used to align the reads to the reference genome, generating an alignment result in the sam text format. The output alignment result is converted into a bam binary format result using the view subroutine of samtools, and the bam file is sorted according to the alignment position using the sort subroutine. Finally, the Mark Duplicates subroutine in the Picard program is used to remove PCR duplicates.

[0142] After obtaining deduplicated bam files, the HaplotypeCaller subroutine of GATK 4.1.4.1 was used to perform genotyping at each locus for each sample, generating a g.vcf file. After obtaining analytical information for each sample, the CombineGVCFs subroutine was used to merge all g.vcf files, and variant detection (including SNPs and indels) was performed using the GenotypeGVCFs subroutine. The variant detection results were extracted using the SelectVariants subroutine, followed by filtering using the VariantFiltration subroutine with parameters set to: quality-by-depth ratio (QD) <2.0 || read mapping quality (MQ) <40.0 || probability of strand bias (FS) >60.0 || symmetric odds ratio (SOR) >3.0 || MQRankSum <-12.5 || ReadPosRankSum <-8.0, completing the initial filtering of low-quality variant sites. Then, vcftools was used to filter SNPs with minor allele frequency less than 1%, variant site missing rate greater than 0%, and allele frequency not equal to 2 to obtain high-quality SNPs for subsequent screening of traceability SNP sites.

[0143] 3. SNP site screening and primer design

[0144] In the present invention, a custom Python script is first used to analyze the population SNP file. Through this script, SNP sites specific to each country can be screened out. Specifically, when samples from a certain country have the same genotype at a certain SNP site, but the genotypes of samples from other countries at this SNP site are different, the SNP is the specific SNP of that country. Next, starting with a certain country (such as the United States), the SNP with the highest quality value among the country-specific SNPs is screened out, and then the samples of that country are removed from the population SNP file. Then, this screening process is repeated with the remaining population SNP files until the last country is screened out for specific SNPs. In this way, a set of SNP sites for national tracing of the fruit fly can be obtained. Annovar is used to annotate the SNP sites, and bedtools is used to extract 300bp of sequences before and after each SNP for subsequent primer design. Based on these sequences, PCR primers can be designed using tools such as Primer 6.0. Through PCR amplification and Sanger sequencing, SNP sites in the fruit fly population can be quickly and accurately detected and the country of origin can be analyzed to achieve the purpose of tracing the source. The SNP site information and primer information are shown in Tables 2 and 3.

[0145] Table 2 Information on SNP combination sites of Bactrocera dorsalis traceability in eight countries

[0146]

[0147] Note: 0 / 0 and 1 / 1 represent homozygous genotypes

[0148] SNP1 is at position 300 of sequence 1 and is either A or C. SNP2 is at position 300 of sequence 2 and is either A or T. SNP3 is at position 300 of sequence 3 and is either G or A. SNP4 is at position 300 of sequence 4 and is either A or G. SNP5 is at position 300 of sequence 5 and is either T or A. SNP6 is at position 300 of sequence 6 and is either A or T. SNP7 is at position 300 of sequence 7 and is either T or C. SNP8 is at position 300 of sequence 8 and is either T or A.

[0149] Sequence 1 is as follows: wherein m is A or C.

[0150] The specific sequence of Sequence 2 is as follows: GAGTTTCAGGCTGAATTTGAAAAAACAACGTTTAGATCGGCCACTTTGTCTTCGCTTTTATGTTGTCTTTGCGAATGTGGTGACGCTTCTGCTCATGCCGGTGGTCTACATCCAGCAGGCGACCGCACTGCAAAAAACTCATGTCGAAAACAGTCTGATCAACTTCACTGATTGTGTGGGTATTATCTGCACATTTACAGCGGCAGCAACTACAGTATTACGCCGTTGGAAGCACGAGGTTCACTATGCTCGCATAGCTCAAGCTATTTTCAAACTCGACAAAGACTACTACAACAAGTwTCCTTGCGACAATGACAAAGACCGACACTCAGACTATTTTCTATACTTCAAGACGCTGACTTTTTCGCTCCATGCAATCGGTGTTTTCTATGGCGCCATTCAGTTCTCGCCCATCTTCAGCTTGAGAGATCTGCTCTTGGCTATATACTAAGGTTATGTATACAACTTGCTTAATGGTTCGCTCTTCATCTATTTCTGCTTCATGTGGCTTTTGCGAAGACGGTTCTCCTTGCTCAATACCAATCTAAACCATTTGCTCCATGCACTGCAGCCGTCGCAAGGGGTCAGTGAGGGCGCATT; where w is A or T.

[0151] The specific sequence of Sequence 3 is as follows: TATAACCGTTCTGTTGTTCTTGTAGCGAGCACAAATGGGAAAGCACTCCATGTAGTGCTTAGTATATATTCGTGACCCGATGTGCGTCATATACCACCCACGTACACCACACACACATACACAAATTACTTGCTTGAGGAGACTTTGTCTGTCTCACACCGAAAACCAACATAGAACTCACGACGATGACGGGTTTATATGTATGTTGGTATGTGTATATATGTATTGTAACTTTGAAGTCGGCAGAAGTAGGGCGCGAATGGGATAAGGAAGAAGCGCAAGTTTATCGCATTCGCTTCrCACATTAAATTTTAAGTCAAGATAAAAATGTTTCGCTCTTCAAGCGCAAACTGCACACGATGTGCGTTGATGAGGAGTTTTTCAACAATCCCACAATTTTGGAGAGCTGCGTCGTGTTGGGCAGACCACCTCGAACAACGCATCATTTCAAGATGTTCGCCACAATATTCTATGCCTGCATTTAATGCTGAAAACCACAAGCGCCAAGCAGTTACTAGTGCGCTAAAAAGCGATCCGAGCACGAAAGGTAGATTCCACAGAATTGAACGCGCTCAACGCTAAGCTACTTCCTTATGTCCG; wherein, r is A or G.

[0152] The specific sequence of Sequence 4 is as follows: TGCCATCTGTATGCCACGACTTAAAATGCTGAGGCGATAAGCTGAAACTTGTTTTCTAAACATGCGTTCTTTAATTTAAATAATTCAACAAATGCTCATATAGAGAGAGAATTGCGAGCTAGTTAAATTTGATATTATTTTAGCAAAATATGTAAATCATATTTTGTGTGCAGTCTTAAAAGTTTTTAATTGCTTTTGTATCTTTAATATTAAAAATATTAAACGCAAGGGCATTAGCAACTTTTTTCGAACTAATATCACATGTATACCTTTCGATTCTCCTCCTTTCTAATTCTTTTrCATACGCATAATTCATTCTCAGCACAGGCTAGAAGTGCCGTGGCCGAGTGCCGGTCACTTGACGCATATGTGCTAGTATTTGCATGGCGCATTGGAGCGACGGCACACAAATATGAGAGTCCCGAAAATCGATTACTTTATAGTTATTGTTATTAATATTATTTCAAGTAATGGTTTCAAGATTTTTTTTTTATAAAGTAACATTCTAAAAATATTTCAAAAGTTTTTAAACGACACACACGAATACGGTCGTTAGATGCCTAATATGTTATAATGAGACAAGTAAGAAAGTGGTAAGTG; wherein, r is A or G.

[0153] The specific sequence of Sequence 5 is as follows: TTCATTTTCACAGATCGAAATATGTTACTACTATAGATGATATTTTTAGTTTTACCTTAACACCACAGATATTCTCCAACCATTGTCCGTCCATAAATACTTATGTATATGTGTACATAATTTTACAATCCACTCAATTTTTTTAATCATAGTCAGCGGCCCTTTTTTCTGCATCTCATACCACCATTTCGTGATCGCAGAATATTCTCGGAATTTTAATAATAATAATCCACGACAACCGCAGTTTGTCGCTGCTGCTAATGAATGAGCAAAGGTGAGGAGGCATGCTTGTACAGCACwTAGCATTGAAAATGCTCATATTTCGTTCACAATGCTTAATACTCCAACGGATCGCGTCGTCTCTCAGCTGTGAGAGAGTGAGTGTGCGTCATATATTTGGGAAGCTTTGTGAAATTCACAACTAATGTATTGTCGTCGCTACCGCTGCTGTAGCTTGGAATGCGGCTGGGTCACTAACACTGCTACCAAATCGAACAGCAACCGCAGCAGCATTGATCACAAACAAGTAATACAACAACAGCAGTCATAACAATCACTGAATCTTACCGACTAAACAAACGAACGAAATACTAGTTTAGC; where w is A or T.

[0154] The specific sequence of Sequence 6 is as follows: TGAAGTATTGCTTGCTGTGTTCTGCTTCTGTTCCCGCTTCATGCACTTGGCTGACGTCAATGAGCTTTTTCATCGATTTTTTACGCTCGTCTGTATGTACCGTTCACTCTCCAATACCGTCCTCACTTGGCAAACAGATGTCCGACTCCTAGAAATTGTAGCATATACATAGACAAAATGCTGGGAAGTTGGGGCGAATACTTAGGCACTTATACAAATACAAATATATCATATAATTATACATATGCATATATGTGTGTATGTAGGTGTAAATATACTGACAGCTTTGATTGCATATTwACTACTTTGTTTTCCAGCGTATATTGTTTGGAGTTGGTGAATATAGTTAACGTTGTTGTAAATATAATTAATATGTTTATGCATAGATATTTGTTGTGACAAACAATTAAATAGCAAATTCTGTAAAGGTAAGAGGATAAAGGTAGTTTAAGTTATTGATATGTTAACGAAAATATTTAGATTAAGACAATCTAAAATGACGACTAAAAATGTTCCACTATCTAAGTAATTATGTAGTATCGTCTAAGTCCGCGTTCATATCCGTTAGAAACAAGCAAGAATTGTACCGCTTTGACAGTG; where w is A or T.

[0155] Sequence 7 is as follows: AGCGTATTTGGTTTCGTTATCTTGCACAAATTTCCGTTTTGATCATTCTTACTAAGGAAGCTTTGGATCTTTTTTTATGCAAAAGATGGAACTTCATGGCACACTTGCGAAAAGAATTTACTTGGGAAAAACGTGTCACCCTAATATACATATACACCCGTACATGCATATATTTGCGTGTGTATGCACAATGAAATGCAATCTAACAAATACGACAGTTGCTTGTATGTATAGGTAAATGCAAAAACAATAATGCAAGTTGTTAGTACAAACACCAGCAACAACAAATAAAATACACAyTGTGCATTGAGTAAGCACTAAGTGGGTGAAAGGATACGCAAAGTAAATGCAAATAAAGTATATTTGAGTTTACCATACTGTAACTACATACATGTAGCTGTGTATATATGTATAACTATTCTATATATAAATGTATGTATGTCAGTGATCCTTATGGCTCTTACACCGCATTAAACAACATACACATGTATGTATATATGTATGTAAATATTTGCATATAAATTTGCCTTGTTTAGCTCTGAACGGCAGGTGAAGGGTGCACTAAAGTGCCAGTAGCTGTAAATACGAAGTGTCTGAGGT; where y is T or C.

[0156] Sequence 8 is as follows: wherein w is A or T.

[0157] Table 3 Primer sequences for amplifying the SNP combination sites of Bactrocera dorsalis in eight countries

[0158]

[0159] SNP1-F and SNP1-R are specific detection primers designed for SNP1, SNP2-F and SNP2-R are specific detection primers designed for SNP2, SNP3-F and SNP3-R are specific detection primers designed for SNP3, SNP4-F and SNP4-R are specific detection primers designed for SNP4, SNP5-F and SNP5-R are specific detection primers designed for SNP5, SNP6-F and SNP6-R are specific detection primers designed for SNP6, SNP7-F and SNP7-R are specific detection primers designed for SNP7, and SNP8-F and SNP8-R are specific detection primers designed for SNP8.

[0160] 3. Application of SNP locus combination in tracing the origin of Bactrocera dorsalis population

[0161] 1. Sample Collection and Genomic DNA Extraction

[0162] 83 oriental fruit flies of known geographical origin were selected (6 samples from Kuala Lumpur, Malaysia, sample number MYKL01-06; 10 samples from Mpumalanga, South Africa, sample number SAMP01-10; 10 samples from Hawaii, USA, sample number USHW01-10; 8 samples from Davao, Philippines, sample number PHDV01-08; 5 samples from Bangkok, Thailand, sample number THBK01-05; 5 samples from Phuket, Thailand, sample number THPK01-05; 3 samples from Tien Giang, Vietnam, sample number VNTG01-03; 5 samples from Guangdong, China) Guangzhou, sample number GDGZ01-05; 5 samples from Zhangzhou, Fujian, China, sample number FJZZ01-05; 5 samples from Yuanjiang, Yunnan, China, sample number YNYJ01-05; 6 samples from Telangana, India, sample number INTG01-06; 5 samples from Murree, Pakistan, sample number PKMR01-05; 5 samples from Port Moresby, Papua New Guinea, sample number PGPM01-05; 5 samples from Vientiane, Laos, sample number LAVT01-05) were used for population tracing and to verify the accuracy of the SNP site combination, as shown in Table 4. The genomic DNA of 83 samples was extracted using the blood / cell / tissue genomic DNA extraction kit (DP304-03) from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the genomic DNA of MYKL01, MYKL02, MYKL03, MYKL04, MYKL05, MYKL06, SAMP01, SAMP02, SAMP03, SAMP04, SAMP05, SAMP06, SAMP07, SAMP08, SAMP09, SAMP10, USHW01, U SHW02 genomic DNA, USHW03 genomic DNA, USHW04 genomic DNA, USHW05 genomic DNA, USHW06 genomic DNA, USHW07 genomic DNA, USHW08 genomic DNA, USHW09 genomic DNA, USHW10 genomic DNA, PHDV01 genomic DNA, PHDV02 genomic DNA, PHDV03 genomic DNA, PHDV04 genomic DNA, PHDV05 genomic DNA, PHDV06 genomic DNA, PHDV07 genomic DNA, PHDV08 genomic DNA, THBK01 genomic DNA, THBK02 genomic DNA, THBK03 genomic DNA, THBK04 genomic DNA, THBK05 genomic DNA,THPK01 genomic DNA, THPK02 genomic DNA, THPK03 genomic DNA, THPK04 genomic DNA, THPK05 genomic DNA, VNTG01 genomic DNA, VNTG02 genomic DNA, VNTG03 genomic DNA, GDGZ01 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, FJZZ01 genomic DNA, FJZZ02 genomic DNA, FJZZ03 genomic DNA, FJZZ04 genomic DNA, FJZZ05 genomic DNA, YNYJ01 genomic DNA, YNYJ02 genomic DNA, YNYJ03 genomic DNA, YNYJ04 genomic DNA, YNYJ05 genomic DNA, INTG01 genomic DNA, INTG02 genomic DNA, INTG03 genomic DNA, INTG04 genomic DNA, INTG05 genomic DNA, INTG06 genomic DNA, PKMR01 genomic DNA, PKMR02 genomic DNA, PKMR03 genomic DNA, PKMR04 genomic DNA, PKMR05 genomic DNA, PGPM01 genomic DNA, PGPM02 genomic DNA, PGPM03 genomic DNA, PGPM04 genomic DNA, PGPM05 genomic DNA, LAVT01 genomic DNA, LAVT02 genomic DNA, LAVT03 genomic DNA, LAVT04 genomic DNA, LAVT05 genomic DNA.

[0163] Table 4 Information on 83 Bactrocera dorsalis samples of known origin

[0164]

[0165] 2. Genotyping of samples and detection of the origin of the citrus fruit fly

[0166] The genomic DNA of 83 samples of Bactrocera dorsalis in Table 4 (MYKL01 genomic DNA, MYKL02 genomic DNA, MYKL03 genomic DNA, MYKL04 genomic DNA, MYKL05 genomic DNA, MYKL06 genomic DNA, SAMP01 genomic DNA, SAMP02 genomic DNA, SAMP03 genomic DNA, SAMP04 genomic DNA, SAMP05 genomic DNA, SAMP06 genomic DNA, SAMP07 genomic DNA, SAMP08 genomic DNA, SAMP09 genomic DNA, SAMP10 genomic DNA, USHW01 genomic DNA) were used respectively. A, USHW02 genomic DNA, USHW03 genomic DNA, USHW04 genomic DNA, USHW05 genomic DNA, USHW06 genomic DNA, USHW07 genomic DNA, USHW08 genomic DNA, USHW09 genomic DNA, USHW10 genomic DNA, PHDV01 genomic DNA, PHDV02 genomic DNA, PHDV03 genomic DNA, PHDV04 genomic DNA, PHDV05 genomic DNA, PHDV06 genomic DNA, PHDV07 genomic DNA, PHDV08 genomic DNA, THBK01 genomic DNA, THBK02 genomic DNA genomic DNA, THBK03 genomic DNA, THBK04 genomic DNA, THBK05 genomic DNA, THPK01 genomic DNA, THPK02 genomic DNA, THPK03 genomic DNA, THPK04 genomic DNA, THPK05 genomic DNA, VNTG01 genomic DNA, VNTG02 genomic DNA, VNTG03 genomic DNA, GDGZ01 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, FJZZ01 genomic DNA, FJZZ02 genomic DNA, FJZ Z03 genomic DNA, FJZZ04 genomic DNA, FJZZ05 genomic DNA, YNYJ01 genomic DNA, YNYJ02 genomic DNA, YNYJ03 genomic DNA, YNYJ04 genomic DNA, YNYJ05 genomic DNA, INTG01 genomic DNA, INTG02 genomic DNA, INTG03 genomic DNA, INTG04 genomic DNA, INTG05 genomic DNA, INTG06 genomic DNA, PKMR01 genomic DNA, PKMR02 genomic DNA, PKMR03 genomic DNA, PKMR04 genomic DNA, PKMR05 genomic DNA,PCR amplification was performed using PGPM01 genomic DNA, PGPM02 genomic DNA, PGPM03 genomic DNA, PGPM04 genomic DNA, PGPM05 genomic DNA, and LAVT01 genomic DNA, LAVT02 genomic DNA, LAVT03 genomic DNA, LAVT04 genomic DNA, and LAVT05 genomic DNA as templates using a traceability SNP primer combination. The traceability SNP primer combination consists of eight primer pairs: SNP1-F / SNP1-R, SNP2-F / SNP2-R, SNP3-F / SNP3-R, SNP4-F / SNP4-R, SNP5-F / SNP5-R, SNP6-F / SNP6-R, SNP7-F / SNP7-R, and SNP8-F / SNP8-R. These eight primer pairs are packaged separately.

[0167] SNP1-F and SNP1-R are specific detection primers designed for SNP1, SNP2-F and SNP2-R are specific detection primers designed for SNP2, SNP3-F and SNP3-R are specific detection primers designed for SNP3, SNP4-F and SNP4-R are specific detection primers designed for SNP4, SNP5-F and SNP5-R are specific detection primers designed for SNP5, SNP6-F and SNP6-R are specific detection primers designed for SNP6, SNP7-F and SNP7-R are specific detection primers designed for SNP7, and SNP8-F and SNP8-R are specific detection primers designed for SNP8. SNP1-F, SNP2-F, SNP3-F, SNP4-F, SNP5-F, SNP6-F, SNP7-F and SNP8-F are upstream primers, and SNP1-R, SNP2-R, SNP3-R, SNP4-R, SNP5-R, SNP6-R, SNP7-R and SNP8-R are downstream primers.

[0168] Each genomic DNA (MYKL01 genomic DNA, MYKL02 genomic DNA, MYKL03 genomic DNA, MYKL04 genomic DNA, MYKL05 genomic DNA, MYKL06 genomic DNA, SAMP01 genomic DNA, SAMP02 genomic DNA, SAMP03 genomic DNA, SAMP04 genomic DNA, SAMP05 genomic DNA, SAMP06 genomic DNA, SAMP07 genomic DNA, SAMP08 genomic DNA, SAMP09 genomic DNA, SAMP10 genomic DNA, USHW01 genomic DNA, USHW02 genomic DNA) DNA, USHW03 genomic DNA, USHW04 genomic DNA, USHW05 genomic DNA, USHW06 genomic DNA, USHW07 genomic DNA, USHW08 genomic DNA, USHW09 genomic DNA, USHW10 genomic DNA, PHDV01 genomic DNA, PHDV02 genomic DNA, PHDV03 genomic DNA, PHDV04 genomic DNA, PHDV05 genomic DNA, PHDV06 genomic DNA, PHDV07 genomic DNA, PHDV08 genomic DNA, THBK01 genomic DNA, THBK02 genomic DNA, BK03 genomic DNA, THBK04 genomic DNA, THBK05 genomic DNA, THPK01 genomic DNA, THPK02 genomic DNA, THPK03 genomic DNA, THPK04 genomic DNA, THPK05 genomic DNA, VNTG01 genomic DNA, VNTG02 genomic DNA, VNTG03 genomic DNA, GDGZ01 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, GDGZ02 genomic DNA, FJZZ01 genomic DNA, FJZZ02 genomic DNA, FJZZ03 genomic DNA genomic DNA, FJZZ04 genomic DNA, FJZZ05 genomic DNA, YNYJ01 genomic DNA, YNYJ02 genomic DNA, YNYJ03 genomic DNA, YNYJ04 genomic DNA, YNYJ05 genomic DNA, INTG01 genomic DNA, INTG02 genomic DNA, INTG03 genomic DNA, INTG04 genomic DNA, INTG05 genomic DNA, INTG06 genomic DNA, PKMR01 genomic DNA, PKMR02 genomic DNA, PKMR03 genomic DNA, PKMR04 genomic DNA, PKMR05 genomic DNA,PGPM01 genomic DNA, PGPM02 genomic DNA, PGPM03 genomic DNA, PGPM04 genomic DNA, PGPM05 genomic DNA, LAVT01 genomic DNA, LAVT02 genomic DNA, LAVT03 genomic DNA, LAVT04 genomic DNA, LAVT05 genomic DNA) were amplified respectively. The PCR reaction systems of the eight primer pairs are shown in Table 5; the PCR amplification conditions of the eight primer pairs are shown in Table 6.

[0169] Table 5 PCR reaction system (25ul)

[0170]

[0171] Table 6 PCR amplification conditions

[0172]

[0173] After PCR amplification, the crude PCR product was directly sent to BGI for purification and Sanger sequencing. Sequencing data were obtained and SNP genotyping was performed using Mega11 and Chromas, yielding the genotypes of eight traceable SNP loci for known Bactrocera dorsalis samples (Table 7).

[0174] Use the following judgment method to make a judgment:

[0175] Each sample is determined in the order of SNP1-SNP8:

[0176] If the genotype of SNP1 is CC, the geographical population of the sample is the United States, and the discrimination ends;

[0177] If the genotype of SNP1 is not CC, the sample is from another geographical population and the determination of SNP2 continues. If the genotype of SNP2 is TT, the geographical population of the sample is the Philippines and the determination ends.

[0178] If the genotype of SNP2 is not TT, the sample is from another geographical population and the determination of SNP3 continues. If the genotype of SNP3 is AA, the geographical population of the sample is South Africa and the determination ends.

[0179] If the genotype of SNP3 is not AA, the sample is from another geographical population and the determination of SNP4 continues. If the genotype of SNP4 is GG, the geographical population of the sample is Vietnam and the determination ends.

[0180] If the genotype of SNP4 is not GG, the sample is from another geographical population and the determination of SNP5 continues. If the genotype of SNP5 is AA, the geographical population of the sample is Malaysia and the determination ends.

[0181] If the genotype of SNP5 is not AA, the sample is from another geographical population and the determination of SNP6 continues. If the genotype of SNP6 is TT, the geographical population of the sample is India and the determination ends.

[0182] If the genotype of SNP6 is not TT, the sample is from another geographical population, and the determination of SNP7 continues. If the genotype of SNP7 is TT, the geographical population of the sample is China, and the determination ends.

[0183] If the genotype of SNP7 is not TT, the sample is from another geographical population, and the determination of SNP8 continues. If the genotype of SNP8 is AA, the geographical population of the sample is Thailand, and the determination ends.

[0184] If the genotype of SNP8 is not AA, the sample is from another geographical population.

[0185] The genotype of SNP1 is CC, which indicates a homozygous C at position 300 of Sequence 1. The genotype of SNP2 is TT, which indicates a homozygous T at position 300 of Sequence 2. The genotype of SNP3 is AA, which indicates a homozygous A at position 300 of Sequence 3. The genotype of SNP4 is GG, which indicates a homozygous G at position 300 of Sequence 4. The genotype of SNP5 is AA, which indicates a homozygous A at position 300 of Sequence 5. The genotype of SNP6 is TT, which indicates a homozygous T at position 300 of Sequence 6. The genotype of SNP7 is CC, which indicates a homozygous C at position 300 of Sequence 7. The genotype of SNP8 is AA, which indicates a homozygous A at position 300 of Sequence 8.

[0186] The genotype of SNP1 is AA, which indicates the homozygous type for A at position 300 of Sequence 1. The genotype of SNP2 is AA, which indicates the homozygous type for A at position 300 of Sequence 2. The genotype of SNP3 is GG, which indicates the homozygous type for G at position 300 of Sequence 3. The genotype of SNP4 is AA, which indicates the homozygous type for A at position 300 of Sequence 4. The genotype of SNP5 is TT, which indicates the homozygous type for T at position 300 of Sequence 5. The genotype of SNP6 is AA, which indicates the homozygous type for A at position 300 of Sequence 6. The genotype of SNP7 is TT, which indicates the homozygous type for T at position 300 of Sequence 7. The genotype of SNP8 is TT, which indicates the homozygous type for T at position 300 of Sequence 8.

[0187] The genotype of SNP1 is AC, which is a heterozygous type with A at position 300 of sequence 1 and C at position 300 of sequence 1. The genotype of SNP2 is AT, which is a heterozygous type with A at position 300 of sequence 2 and T at position 300 of sequence 2. The genotype of SNP3 is GA, which is a heterozygous type with G at position 300 of sequence 3 and A at position 300 of sequence 3. The genotype of SNP4 is AG, which is a heterozygous type with A at position 300 of sequence 4 and G at position 300 of sequence 4. The genotype of SNP5 is TA, which is a heterozygous type with T at position 300 of sequence 5 and A at position 300 of sequence 5. The genotype of SNP6 is AT, which is a heterozygous type with A at position 300 of sequence 6 and T at position 300 of sequence 6. The genotype of SNP7 is TC, which is a heterozygous type with T at position 300 of sequence 7 and C at position 300 of sequence 7. The genotype of SNP8 is TA, which is a heterozygous type in which the 300th position of sequence 8 is T and the 300th position of sequence 8 is A.

[0188] The test results in Table 7 indicate that the 68 sample collection locations within the target country of origin are consistent with the country of origin traceability results obtained using the SNP locus combination of the present invention. Therefore, the SNP locus combination of the present invention can be used to detect the genotype of the citrus fruit fly to achieve the purpose of tracing the source of some countries.

[0189] The present invention provides a SNP site and detection primer combination and application for national-level tracing of the citrus fruit fly. The method is simple to operate, short in time, low in cost, highly polymorphic and stable, and is applicable to different populations of the citrus fruit fly. The application of the present invention will contribute to the monitoring of the citrus fruit fly population and the development of pest and disease control work. Through the application of this patented technology, the country of origin of the citrus fruit fly can be quickly and accurately analyzed and traced, providing a scientific basis for pest monitoring. It helps to prevent and manage the invasion and spread of foreign pests such as the citrus fruit fly. Timely and effective prevention of biological invasions is of great significance for maintaining national biosecurity.

[0190] Table 7 Genotyping of 8 traceable SNP loci of known samples of Bactrocera dorsalis

[0191]

[0192]

[0193]

[0194]

[0195] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Application, characterized by: The application is any of the following: 1) Use of a substance for detecting the SNP1 genotype, a substance for detecting the SNP2 genotype, a substance for detecting the SNP3 genotype, a substance for detecting the SNP4 genotype, a substance for detecting the SNP5 genotype, a substance for detecting the SNP6 genotype, a substance for detecting the SNP7 genotype, and a substance for detecting the SNP8 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from Thailand; 2) Use of substances for detecting the SNP1 genotype, substances for detecting the SNP2 genotype, substances for detecting the SNP3 genotype, substances for detecting the SNP4 genotype, substances for detecting the SNP5 genotype, substances for detecting the SNP6 genotype, and substances for detecting the SNP7 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from China; 3) Use of a substance for detecting the SNP1 genotype, a substance for detecting the SNP2 genotype, a substance for detecting the SNP3 genotype, a substance for detecting the SNP4 genotype, a substance for detecting the SNP5 genotype, and a substance for detecting the SNP6 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from India; 4) Use of a substance for detecting the SNP1 genotype, a substance for detecting the SNP2 genotype, a substance for detecting the SNP3 genotype, a substance for detecting the SNP4 genotype, and a substance for detecting the SNP5 genotype in identifying or assisting in identifying whether a fruit fly to be tested is a product originating from Malaysia; 5) Use of substances for detecting the SNP1 genotype, substances for detecting the SNP2 genotype, substances for detecting the SNP3 genotype, and substances for detecting the SNP4 genotype in identifying or assisting in identifying whether a product containing fruit flies to be tested originates from Vietnam; 6) Use of substances for detecting the SNP1 genotype, substances for detecting the SNP2 genotype, and substances for detecting the SNP3 genotype in identifying or assisting in identifying whether a fruit fly species to be tested is a product originating from South Africa; 7) Use of substances for detecting the SNP1 genotype and substances for detecting the SNP2 genotype in identifying or assisting in identifying whether a product containing fruit flies originates from the Philippines; 8) Use of substances for detecting the SNP1 genotype in identifying or assisting in identifying whether a fruit fly species being tested is a product originating from the United States; The SNP1 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 1 and is A or C; The SNP2 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 2 and is A or T; The SNP3 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 3 and is G or A; The SNP4 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 4 and is A or G; The SNP5 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 5, and is T or A; The SNP6 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 6, and is A or T; The SNP7 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 7, which is T or C; The SNP8 is a SNP in the fruit fly genome, which is the 300th nucleotide of sequence 8 and is T or A.

2. The use according to claim 1, characterized in that The fruit fly to be tested is Bactrocera dorsalis.

3. The method, characterized in that The method is any of the following: M1) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from Thailand, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from Thailand based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, SNP5 genotype, SNP6 genotype, SNP7 genotype, and SNP8 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, the SNP7 genotype is not TT, and the SNP8 genotype is AA, the fruit fly to be tested is from Thailand or the candidate is from Thailand; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, the SNP7 genotype is not TT, and the SNP8 genotype is not AA, the fruit fly to be tested is not from Thailand or the candidate is not from Thailand; M2) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from China, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from China based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, SNP5 genotype, SNP6 genotype, and SNP7 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, and the SNP7 genotype is TT, the fruit fly to be tested is from China or the candidate is from China; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, the SNP6 genotype is not TT, and the SNP7 genotype is not TT, the fruit fly to be tested is not from China or the candidate is not from China; M3) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from India, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from India based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, SNP5 genotype, and SNP6 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, and the SNP6 genotype is TT, the fruit fly to be tested is from India or the candidate is from India; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, the SNP5 genotype is not AA, and the SNP6 genotype is not TT, the fruit fly to be tested is not from India or the candidate is not from India; M4) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from Malaysia, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from Malaysia based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, SNP4 genotype, and SNP5 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, and the SNP5 genotype is AA, the fruit fly to be tested is from Malaysia or the candidate is from Malaysia; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, the SNP4 genotype is not GG, and the SNP5 genotype is not AA, the fruit fly to be tested is not from Malaysia or the candidate is not from Malaysia; M5) a method for identifying or assisting in identifying whether a test fruit fly originates from Vietnam, comprising detecting a SNP genotype of the test fruit fly, and determining whether the test fruit fly originates from Vietnam based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, SNP3 genotype, and SNP4 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, and the SNP4 genotype is GG, the fruit fly to be tested is from Vietnam or the candidate is from Vietnam; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, the SNP3 genotype is not AA, and the SNP4 genotype is not GG, the fruit fly to be tested is not from Vietnam or the candidate is not from Vietnam; M6) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from South Africa, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from South Africa based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype, SNP2 genotype, and SNP3 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, and the SNP3 genotype is AA, the fruit fly to be tested is from South Africa or the candidate is from South Africa; if the SNP1 genotype of the fruit fly to be tested is not CC, the SNP2 genotype is not TT, and the SNP3 genotype is not AA, the fruit fly to be tested is not from South Africa or the candidate is not from South Africa; M7) a method for identifying or assisting in identifying whether a fruit fly to be tested originates from the Philippines, comprising detecting a SNP genotype of the fruit fly to be tested, and determining whether the fruit fly to be tested originates from the Philippines based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype and the SNP2 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is not CC and the SNP2 genotype is TT, the fruit fly to be tested is from the Philippines or the candidate is from the Philippines; if the SNP1 genotype of the fruit fly to be tested is not CC and the SNP2 genotype is not TT, the fruit fly to be tested is not from the Philippines or the candidate is not from the Philippines; M8) A method for identifying or assisting in identifying whether a test fruit fly originates from the United States, comprising detecting a SNP genotype of the test fruit fly, and determining whether the test fruit fly originates from the United States based on the genotype detection result, wherein the SNP genotype is the SNP1 genotype described in claim 1; If the SNP1 genotype of the fruit fly to be tested is CC, the fruit fly to be tested is from the United States or the candidate is from the United States; if the SNP1 genotype of the fruit fly to be tested is not CC, the fruit fly to be tested is not from the United States or the candidate is not from the United States; The genotype of SNP1 is CC, which is a homozygous type with C at position 300 of sequence 1; The genotype of SNP2 is TT, which is a homozygous type with T at position 300 of sequence 2; The genotype of SNP3 is AA, which is a homozygous type with A at position 300 of sequence 3; The genotype of SNP4 is GG, which is a homozygous type with G at position 300 of sequence 4; The genotype of SNP5 is AA, which is a homozygous type with A at position 300 of sequence 5; The genotype of SNP6 is TT, which is a homozygous type with T at position 300 of sequence 6; The genotype of SNP7 is TT, which is a homozygous type with T at position 300 of sequence 7; The genotype of SNP8 is AA, which is a homozygous type in which the 300th position of sequence 8 is A.

4. Use of the method according to claim 3 in identifying or assisting in identifying the national geographical origin of Tephritidae animals.

5. The product is characterized in that The product is any of the following: P1) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from Thailand, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 as recited in claim 1; P2) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from China, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7 as recited in claim 1; P3) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from India, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, SNP5, and SNP6 as recited in claim 1; P4) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from Malaysia, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, SNP4, and SNP5 as recited in claim 1; P5) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from Vietnam, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, SNP3, and SNP4 as recited in claim 1; P6) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from South Africa, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1, SNP2, and SNP3 as described in claim 1; P7) identifying or assisting in identifying whether the fruit fly to be tested is a product originating from the Philippines, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is SNP1 and SNP2 as described in claim 1; P8) Identifying or assisting in identifying whether the fruit fly to be tested is a product originating from the United States, comprising a substance for detecting the genotype of a SNP of the fruit fly to be tested, wherein the SNP is the SNP1 described in claim 1.

6. The use according to claim 1 or 2, the method according to claim 3, the use according to claim 4, or the product according to claim 5, characterized in that: The products of the substances for detecting the genotypes of the eight SNPs SNP1-SNP8 are the following D1), D2), D3) or D4): D1) containing in vitro nucleic acid amplification primers for specifically amplifying and detecting the SNP; D2) an in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in D1); D3) a kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2); D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2) or the kit described in D3).

7. The use, product or method according to claim 6, characterized in that The in vitro nucleic acid amplification primers include a primer pair SNP1-F / SNP1-R for amplifying SNP1, a primer pair SNP2-F / SNP2-R for amplifying SNP2, a primer pair SNP3-F / SNP3-R for amplifying SNP3, a primer pair SNP4-F / SNP4-R for amplifying SNP4, a primer pair SNP5-F / SNP5-R for amplifying SNP5, a primer pair SNP6-F / SNP6-R for amplifying SNP6, a primer pair SNP7-F / SNP7-R for amplifying SNP7, and a primer pair SNP8-F / SNP8-R for amplifying SNP8; The SNP1-F sequence is shown in Sequence 9; the SNP1-R sequence is shown in Sequence 10; the SNP2-F sequence is shown in Sequence 11; the SNP2-R sequence is shown in Sequence 12; the SNP3-F sequence is shown in Sequence 13; the SNP3-R sequence is shown in Sequence 14; the SNP4-F sequence is shown in Sequence 15; the SNP4-R sequence is shown in Sequence 16; the SNP5-F sequence is shown in Sequence 17; the SNP5-R sequence is shown in Sequence 18; the SNP6-F sequence is shown in Sequence 19; the SNP6-R sequence is shown in Sequence 20; the SNP7-F sequence is shown in Sequence 21; the SNP7-R sequence is shown in Sequence 22; the SNP8-F sequence is shown in Sequence 23; and the SNP8-R sequence is shown in Sequence 24.

8. DNA barcode of Bactrocera dorsalis, characterized in that: The DNA barcodes are the 8 SNPs described in claim 1.

9. Use of the DNA barcode according to claim 8 in identifying or assisting in identifying the national geographical origin of Tephritidae animals, or in preparing a substance for identifying or assisting in identifying the national geographical origin of Tephritidae animals.

10. A composition characterized in that The composition contains one or more of a substance for detecting SNP1 genotype, a substance for detecting SNP2 genotype, a substance for detecting SNP3 genotype, a substance for detecting SNP4 genotype, a substance for detecting SNP5 genotype, a substance for detecting SNP6 genotype, a substance for detecting SNP7 genotype and a substance for detecting SNP8 genotype.

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