Application of miRNA-SSR molecular marker primers in identification of heat tolerance in grapes

Abstract

The present invention discloses the application of grape miRNA-SSR molecular marker primers in the identification of heat resistance of grapes. The grape miRNA-SSR molecular marker primers are selected from the group formed by the following primers: VMIRSSR156c1, VMIRSSR157c2, VMIRSSR159f2, VMIRSSR160c1, VMIRSSR164b1, VMIRSSR167c3, VMIRSSR169d1, VMIRSSR171b1, VMIRSSR171k1, VMIRSSR398b1, VMIRSSR398c3, VMIRSSR167d1 and VMIRSSR828a1. The miRNA-SSR-based screening method of the present invention provides an important basis for the identification of heat resistance of grape varieties and molecular marker-assisted breeding, enriches the types of molecular marker identification, and improves the identification efficiency.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of grape miRNA-SSR molecular marker primers in identifying heat resistance of grapes. Background Art

[0002] Grape (Vitis vinifera spp.) is a deciduous vine plant of the genus Vitis in the family Vitaceae. It has a long history of cultivation. The grape industry has become an important part of my country's fruit industry. However, while the grape industry is developing rapidly, it is also facing severe challenges. In recent years, with the intensification of the greenhouse effect, global temperatures have continued to rise. According to the fifth assessment report of the Intergovernmental Panel on Climate Change (IPCC), by 2100, global temperatures will even increase by 1-3°C. In addition, the main grape varieties planted in my country are Eurasian and European and American hybrid grapes, which have poor tolerance to high temperature stress. High temperature can cause the leaves of grapes to curl, lose water and dry up, reduce photosynthetic efficiency, and have poor fruit quality, which in turn affects physiological and biochemical reactions and the expression and regulation of adversity-induced genes, seriously damages the ability of self-adversity regulation, causes heat damage and even wilts and dies, and largely restricts the development of the grape industry. Chinese researchers have long noticed the problem of high temperature resistance of grapes and have carried out targeted research. However, with the increase in the cultivation area of ​​Eurasian grapes, the heat damage problems in production are also increasing, mainly manifested in poor growth and development of leaves and branches, and reduced fruit quality. At present, little is known about high-quality grape resources that can resist high temperatures, and the development of molecular markers is relatively simple, resulting in low breeding efficiency. Therefore, the development of molecular markers and the identification and breeding of heat-resistant varieties have become important directions for grape stress-resistant breeding.

[0003] At present, molecular markers have been widely used in germplasm resource research, genetic map construction, genetic diversity analysis, kinship research, target gene positioning and auxiliary marker selection breeding. Microsatellite is a simple repeat sequence (SSR) formed by 2-6 nucleotide tandem repeat fragments and evenly distributed in the genome. It has the advantages of high polymorphism, strong stability, simple operation, multiple alleles, abundant quantity and good genome coverage, and has become the main technology for variety identification. By analyzing the SSRs existing in the reference genome of the species, polymerase chain reaction (PCR) combined with agarose gel electrophoresis, polyvinyl chloride gel electrophoresis and other detection and analysis are further used, and finally combined with biological statistical analysis to obtain the polymorphic allele value detected by the molecular marker. Abroad, 9 pairs of SSR primers such as VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG79 and VrZAG62 have been used as international universal markers for grape variety identification. As an important gene expression regulatory factor, miRNA plays a critical role in plant growth and development and stress. Studies have shown that multiple miRNAs play an important role in the growth and development of grapes, such as miR164b and miR828; and it was found that the polypeptide Vvi-miPEP171d1 encoded by the primary transcript of miR171d, pri-miR171d, can promote the formation of adventitious roots in grape tissue culture seedlings. However, there are no reports on SSR molecular markers in the pri-miRNA nucleotide sequence in grapes. In previous studies, the verification of SSR polymorphism mostly used various biological statistics of electrophoresis-running gel results. This method is time-consuming and labor-intensive, and the pollution to the laboratory is also serious. In addition, the difference in bands in the gel image may not be due to the difference in SSR repeat types, but is likely to be caused by the absence of some sequences, which largely indicates that the experimental data and analysis results are inaccurate. Therefore, the development of miRNA-SSR molecular markers based on grape reference genome data provides important evidence for the identification of heat-resistant grape varieties and molecular marker-assisted breeding. Summary of the invention

[0004] One of the purposes of the present invention is to provide a grape miRNA-SSR molecular marker primer.

[0005] The second object of the present invention is to provide the application of the above-mentioned grape miRNA-SSR molecular marker primers in the identification of heat resistance of grape varieties.

[0006] A third object of the present invention is to provide a kit for identifying the heat resistance of grapes.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The grape miRNA-SSR molecular marker primers provided by the present invention are selected from the group formed by the following primers for detecting SSR diversity: VMIRSSR156c1, VMIRSSR157c2, VMIRSSR159f2, VMIRSSR160c1, VMIRSSR164b1, VMIRSSR167c3, VMIRSSR169d1, VMIRSSR171b1, VMIRSSR171k1, VMIRSSR398b1, VMIRSSR398c3, VMIRSSR167d1 and VMIRSSR828a1; wherein:

[0009] The forward and reverse primer sequences of VMIRSSR156c1 are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0010] The forward and reverse primer sequences of VMIRSSR157c2 are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0011] The forward and reverse primer sequences of VMIRSSR159f2 are shown in SEQ ID NO.5 and SEQ ID NO.6;

[0012] The forward and reverse primer sequences of VMIRSSR160c1 are shown in SEQ ID NO.7 and SEQ ID NO.8;

[0013] The forward and reverse primer sequences of VMIRSSR164b1 are shown in SEQ ID NO.9 and SEQ ID NO.10;

[0014] The forward and reverse primer sequences of VMIRSSR167c3 are shown in SEQ ID NO.11 and SEQ ID NO.12;

[0015] The forward and reverse primer sequences of VMIRSSR169d1 are shown in SEQ ID NO.13 and SEQ ID NO.14;

[0016] The forward and reverse primer sequences of VMIRSSR171b1 are shown in SEQ ID NO.15 and SEQ ID NO.16;

[0017] The forward and reverse primer sequences of VMIRSSR171k1 are shown in SEQ ID NO. 17 and SEQ ID NO. 18;

[0018] The forward and reverse primer sequences of VMIRSSR398b1 are shown in SEQ ID NO.19 and SEQ ID NO.20;

[0019] The forward and reverse primer sequences of VMIRSSR398c3 are shown in SEQ ID NO.21 and SEQ ID NO.22;

[0020] The forward and reverse primer sequences of VMIRSSR167d1 are shown in SEQ ID NO.23 and SEQ ID NO.24;

[0021] The forward and reverse primer sequences of VMIRSSR828a1 are shown in SEQ ID NO.25 and SEQ ID NO.26.

[0022] The present invention also provides application of the grape miRNA-SSR molecular marker primers in identifying the heat resistance of grape varieties.

[0023] Preferably, the grape miRNA-SSR molecular marker primers are selected from the group consisting of the following primers: VMIRSSR156c1, VMIRSSR157c2, VMIRSSR159f2, VMIRSSR167c3, VMIRSSR171b1, VMIRSSR171k1, VMIRSSR398b1 and VMIRSSR167d1.

[0024] Preferably, the grape varieties are shown in the following table:

[0025]

[0026]

[0027] As a more preferred embodiment, the grape varieties are shown in the following table:

[0028] NO. variety Economic Value type source 1 Champagne grapes Wine grapes AM Zhengzhou 2 Shen Mei Table grapes V L Shanghai 3 Xinya Wine grapes VI Xinjiang 4 Double Red Wine grapes AW Beijing 5 Beida Rootstock grape AM USA 6 Niagara Rosada Wine grapes V L Shanghai 7 Cabernet Sauvignon Wine grapes VI France 8 Seedless White Table grapes VI Xinjiang

[0029] The present invention also provides the use of grape miRNA-SSR molecular marker primers in the identification of heat-resistant grape varieties, wherein the grape miRNA-SSR molecular marker primers are selected from the group formed by the following primers: VMIRSSR167c3, VMIRSSR167d1, VMIRSSR157c2 and VMIRSSR398b1, wherein:

[0030] The forward and reverse primer sequences of VMIRSSR167c3 are shown in SEQ ID NO.11 and SEQ ID NO.12;

[0031] The forward and reverse primer sequences of VMIRSSR167d1 are shown in SEQ ID NO.23 and SEQ ID NO.24;

[0032] The forward and reverse primer sequences of VMIRSSR157c2 are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0033] The forward and reverse primer sequences of VMIRSSR398b1 are shown in SEQ ID NO.19 and SEQ ID NO.20.

[0034] The present invention also provides the use of grape miRNA-SSR molecular marker primers in the identification of sensitive grape varieties, wherein the grape miRNA-SSR molecular marker primers are selected from the group formed by the following primers: VMIRSSR167c3, VMIRSSR167d1, VMIRSSR157c2 and VMIRSSR398b1, wherein:

[0035] The forward and reverse primer sequences of VMIRSSR167c3 are shown in SEQ ID NO.11 and SEQ ID NO.12;

[0036] The forward and reverse primer sequences of VMIRSSR167d1 are shown in SEQ ID NO.23 and SEQ ID NO.24;

[0037] The forward and reverse primer sequences of VMIRSSR157c2 are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0038] The forward and reverse primer sequences of VMIRSSR398b1 are shown in SEQ ID NO.19 and SEQ ID NO.20.

[0039] In the above technical scheme, the 13 pairs of miRNA-SSR primers and the four heat-resistant and four sensitive varieties screened out in the literature were screened by PCR-agarose gel electrophoresis-high-throughput sequencing, and finally eight pairs of primers were screened out that were polymorphic in varieties with known differences in heat resistance. The 40 grape varieties identified in the early stage (20 sensitive types and 20 heat-resistant types) were selected to distinguish and identify the four pairs of molecular marker primers that met the early heat resistance difference classification.

[0040] Preferably, the grape miRNA-SSR molecular marker primer is VMIRSSR167c3, and its forward and reverse primer sequences are shown in SEQ ID NO.11 and SEQ ID NO.12. A pair of primers VMIRSSR167c3 with a high heat-resistant-sensitive type differentiation rate is selected from 13 pairs of primers, and 18 of 20 heat-resistant varieties and 16 of 20 sensitive varieties can be identified, with differentiation rates of 90% and 80%, respectively.

[0041] The present invention also provides a kit for identifying the heat resistance classification of grapes, which comprises grape miRNA-SSR molecular marker primer VMIRSSR167c3, and the forward and reverse primer sequences thereof are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0042] Compared with the prior art, the present invention has the following beneficial effects: the present invention independently develops grape miRNA-SSR molecular marker primers through the information of the precursor sequence (pre-miRNA) and the primary transcript sequence pri-miRNA (500 bp upstream and downstream of pre-miRNA, about 1000 bp) of miRNA in the reference genome data of Pinot Noir grapes, extracts DNA from heat-resistant and sensitive grape varieties (8 grape varieties), performs PCR detection-agarose gel electrophoresis-sequencing, and screens according to the nucleotide sequence comparison results. Eight pairs of primers with SSR polymorphism (VMIRSSR156c1, VMIRSSR157c2, VMIRSSR159f2, VMIRSSR167c3, VMIRSSR171b1, VMIRSSR171k1, VMIRSSR398b1 and VMIRSSR167d1) were identified, and four pairs of primers (VMIRSSR167c3, VMIRSSR167d1, VMIRSSR157c2 and VMIRSSR398b1) were further selected according to the different heat resistance. Using 40 germplasm resources (20 sensitive and 20 heat-resistant types) that had been identified for heat resistance in the early stage, we carried out detection and analysis of 4 pairs of primer combinations, and screened out the optimal molecular marker VMIRSSR167c3, which has a differentiation rate of 90% and 80% for heat-resistant and sensitive types, respectively. The miRNA-SSR-based screening method provides an important basis for the identification of heat resistance of grape varieties and molecular marker-assisted breeding, enriches the types of molecular marker identification of grape varieties, and improves the identification efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is the growth status of 40 grape germplasm resources in the embodiment.

[0044] Figure 2 It is the sequence comparison result of multiple grape varieties based on SSR in the embodiment. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0046] Example 1

[0047] This example uses 40 grape genotypes of leaf growth and development periods in 2021 preserved in the glass greenhouse of the College of Agriculture and Biology of Shanghai Jiao Tong University. Each variety has 20 grape seedlings, totaling 800 plants. The experimental materials are subject to standard pest, disease control and horticultural practices. The genotype names of these varieties can be divided into VL: V.vinifera×V.labrusca (European and American hybrid), VI: V.vinifera L. (Eurasian species), VA: AV.vinifera×V.amurensis (mountain European species), AM: American wild grapes (American species), AW: Asian wild grapes (Asian species). For detailed information on the annotations and growth conditions of these grape varieties, please refer to Tables 1 and Figure 1 .

[0048] Table 1: VSSRMIRNA marker primer information

[0049]

[0050] Table 2: Species and sources of 40 grape germplasm resources

[0051] Number name Economic Value type source P1 Niagara Rosada Wine grapes V L China P2 Red Ruibao Table grapes V L China P3 Champagne grapes Wine grapes AM China P4 Crisp Red Wine grapes AM China P5 Baisabil Wine grapes V L China P6 Lot Sandy Grapes Wine grapes AM China P7 Da Mpo Wine grapes V L China P8 Aromatic Rachaki Table grapes VI China P9 Pollux Table grapes V L China P10 Aniski Wine grapes V L USA P11 Green beads Table grapes V L China P12 August Table grapes VI Romania P13 Jifeng Table grapes V L China P14 Hegland Wine grapes VI France P15 Shen Mei Table grapes V L China P16 Jiangsu Artemisia Wine grapes AW China P17 Shen Yue Table grapes V L China P18 Shenhua Table grapes VI China P19 Cabernet Sauvignon Wine grapes VI France P20 Volgodon Wine grapes VI Soviet Union P21 Xinya Wine grapes VI China P22 Beichun Wine grapes VA China P23 Jasmine Table grapes V L China P24 Smoke 74 Table grapes VI China P25 Sunshine Rose Table grapes V L Japan P26 White Fuji Table grapes VI Japan P27 Fujiminori Table grapes V L Japan P28 Italy Table grapes VI Hungary P29 SO4 Rootstock AM Germany P30 Seedless Cuibao Table grapes VI China P31 jeloudevyi Table grapes VI China P32 Giant Rose Table grapes V L China P33 Jufeng Table grapes V L Japan P34 Rose scent Table grapes VI U.K. P35 Seedless White Table grapes VI China P36 Double Red Wine grapes AW China P37 1103P Rootstock AM Italy P38 Shenbao Table grapes V L China P39 White Saint Peter Table grapes V L Japan P40 Beida Rootstock AM USA

[0052] 1. DNA extraction method

[0053] The genomic DNA was extracted by the modified CTAB method and used for PCR-electrophoresis-sequencing analysis of miRNA-SSR. The DNA quality was detected by 1% agarose gel electrophoresis. The DNA concentration was determined by Nano drop ultra-micro spectrophotometer and diluted to 50 ng / μL and stored in a -20°C refrigerator for future use.

[0054] 2. SSR primer development

[0055] Based on the results of the Pinot Noir grape reference genome annotation, the grape miRNA precursor sequence (pre-miRNA) and primary transcript sequence pri-miRNA (500 bp upstream and downstream of pre-miRNA, about 1000 bp in total) were obtained, and the SSR polymorphism was predicted by the MISA tool to carry out the prediction statistics of miRNA-SSR. Primer 5.0 software was used to design SSR primers, and 13 pairs of developed SSR primers were selected for PCR amplification and sequencing (Table 2). The primers with polymorphic SSRs screened from 8 varieties were combined with the previous research on high temperature response miRNA to screen out 4 pairs of marker primers (VMIRSSR167c3 (CT), VMIRSSR167d1 (TTTTCC), VMIRSSR157c2 (AGGG) and VMIRSSR398b1 (AGGG)) for heat resistance identification of 40 varieties. The primers were synthesized by Ruimian Biotechnology Co., Ltd.

[0056] 3. PCR amplification

[0057] The PCR amplification system was 30 μL: 50 ng / μL DNA template, 2×TSINGKE Master MIX (blue) 15 μL, 0.5 μL of each upstream (downstream) primer, and the rest was supplemented to 30 μL with dd H2O. The PCR reaction was carried out on a BoLe PCR instrument, and the reaction program was: 95℃5min; 95℃45s, 58-62℃45s, 72℃45s, a total of 34 cycles; 72℃5min; 4℃ storage, and the PCR products were screened for clear products using 1.2% agarose gel electrophoresis bands.

[0058] 4. Sequencing analysis

[0059] 20 μL of PCR product was selected for high-throughput sequencing (completed by Beijing Qingke Biotechnology Co., Ltd.), and DNAMAN software was used for base sequence alignment to further screen the SSR repeat number polymorphism.

[0060] result

[0061] (1) Repeatability analysis of miRNA-SSR molecular markers

[0062] DNA quality was tested by 1% agarose gel electrophoresis and DNA concentration was determined by Nanodrop ultra-micro spectrophotometer. DNA of 40 varieties met the requirements of subsequent experiments. Eight grape varieties (four heat-resistant and four heat-intolerant) were randomly selected to verify the polymorphism of the 13 pairs of SSR primers developed and to perform high-throughput sequencing and comparison analysis. The DNAMAN comparison results are shown in Figure 1, the types of SSR polymorphisms were VMIRSSR156c1, VMIRSSR157c2, VMIRSSR159f2, VMIRSSR167c3, VMIRSSR171b1, VMIRSSR171k1, VMIRSSR398b1 and VMIRSSR167d1, and a total of 8 pairs of polymorphic primers with accurate SSRs in multiple varieties were obtained. Through the joint analysis of the accurate presence of miRNA and SSRs that responded to heat stress in the early screening, 4 pairs of primers were selected for PCR amplification and high-throughput sequencing of genomic DNA of 40 varieties. The statistical differences of different repeatability of SSRs in multiple varieties are shown in Tables 3 and 4.

[0063] (2) VSSRMIR164c3 marker distinguishes between heat-resistant and sensitive types

[0064] Based on the results of the previous physiological data analysis of grape heat resistance and sensitivity, the SSR repetition number of the VSSRMIR164c3 marker in heat-resistant and sensitive grape varieties was further verified. The SSR (CT) repetition number greater than or equal to 18 was defined as a heat-resistant variety, and vice versa, it was defined as a sensitive variety. Biological statistical analysis showed that the differentiation rates of heat-resistant and sensitive types of this combination reached 90% (Table 3) and 80% (Table 4), respectively.

[0065] Table 3: Identification of VMIRSSR167c3, VMIRSSR167d1, VMIRSSR157c2 and VMIRSSR398b1 in heat-tolerant grape varieties

[0066]

[0067] Table 4: Identification of VMIRSSR167c3, VMIRSSR167d1, VMIRSSR157c2 and VMIRSSR398b1 in sensitive grape varieties

[0068]

[0069]

[0070] The above description is only a preferred implementation example of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are within the protection scope of the present invention. Sequence Listing <110> Shanghai Jiao Tong University <120> Application of grape miRNA-SSR molecular marker primers in identification of heat tolerance in grape <141> 2022-06-17 <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence() <400> 1 tgagtgaact tgaggtgcct gcta 24 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence() <400> 2 tgtatggggag ttacaaatgg taaggaata 29 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence() <400> 3 taagtggaag ccctaaggaa gaagaaaaa 29 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence() <400> 4 gaaagaggaa ggcactggtg atgttg 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence() <400> 5 ccgctacatt agttgttata atatcgt 27 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence() <400> 6 cttcttgact tgtaatttgg tttga 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence() <400> 7 aggcataaag ggaaagagga ttggt 25 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence() <400> 8 gtggcgtccg tggaaactga tat 23 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence() <400> 9 taagccaaac agaagctact gtaagt 26 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence() <400> 10 tgaccatctc aacccaataa tacttt 26 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence() <400> 11 tcttcccctt gtataattta ttatgttg 28 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence() <400> 12 catgatctga tctttccttg acttg 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence() <400> 13 aggttgatga gggtgatggt ggttt 25 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence() <400> 14 ctagtgaatg atattaagtt tatgctggta 30 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence() <400> 15 aacctccttc tgctttatcttcccaca 26 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence() <400> 16 gagggaaatc tgctcatctc tcaaactaa 29 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence() <400> 17 attcccattt cttctcacca ctatc 25 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence() <400> 18 gagccaacat cacagtaact atttca 26 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence() <400> 19 aaagtggata tggtaccgaa aagtgatt 28 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence() <400> 20 tagttctgag ggagaagctg gatgac 26 <210> twenty one <211> 26 <212> DNA <213> Artificial Sequence() <400> twenty one acggatgttc aacattaatt tggaag 26 <210> twenty two <211> 27 <212> DNA <213> Artificial Sequence() <400> twenty two ggcatatttt tgagctttat atagggg 27 <210> twenty three <211> 28 <212> DNA <213> Artificial Sequence() <400> twenty three tatcaacctc tctcatgttc ttagggtt 28 <210> twenty four <211> 25 <212> DNA <213> Artificial Sequence() <400> twenty four actaggtaag gagcttaggg caagg 25 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence() <400> 25 aattaacaag aaaaaaaggg gggaaaa 27 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence() <400> 26 agaaagagta agtgagggaa aagggaga 28

Claims

1. A grape miRNA-SSR molecular marker primer, which is VMIRSSR167c3. The forward and reverse primer sequences of VMIRSSR167c3 are shown in SEQ ID NO.11 and SEQ ID NO.

12.

2. Application of the grape miRNA-SSR molecular marker primers according to claim 1 in the identification of heat resistance of grape varieties, wherein the grape varieties are shown in the following table: The VL refers to vinifera×V.labrusca, a European-American hybrid; the VI refers to V.vinifera L., a Eurasian species; the VA refers to AV.vinifera×V.amurensis, a mountain European species; the AM refers to American wild grapes, an American species; and the AW refers to Asian wild grapes, an Asian species.

3. A kit for identifying the heat tolerance classification of grapes, comprising grape miRNA-SSR molecular marker primer VMIRSSR167c3, wherein the forward and reverse primer sequences of VMIRSSR167c3 are shown as SEQ ID NO.11 and SEQ ID NO.12.

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