High-temperature tolerance SNP molecular marker of neoseiulus barkeri as well as application and detection method of high-temperature tolerance SNP molecular marker

Abstract

The invention relates to the technical field of molecular biology, in particular to a high-temperature tolerance SNP (Single Nucleotide Polymorphism) molecular marker of neoseiulus barkeri as well as application and a detection method thereof. The SNP molecular marker is located on at least one of the following sites of a chromosome 2 of a neoseiulus barkeri genome: G / A base mutation exists at 27th, 121th and 501bp sites; g / T base mutation exists at the 27th bp site, the 174th bp site and the 672bp site; c / T base mutation exists at the 27th bp site, the 250th bp site and the 404bp site; t / C base mutation exists at the 27th bp site, the 184th bp site and the 864th bp site. The neoseiulus barkeri high-temperature-resistant related SNP molecular marker provided by the invention has remarkable correlation with a normal-temperature strain and a high-temperature-resistant strain, and the normal-temperature strain and the high-temperature-resistant strain of the neoseiulus barkeri can be quickly identified by detecting sites of the SNP molecular marker; the blank in the field of classification of the neoseiulus barkeri normal-temperature strain and the high-temperature-resistant strain is filled up, and potential application value is achieved for large-scale molecular breeding of the neoseiulus barkeri.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly to SNP molecular markers for high-temperature tolerance of Neoseiulus barkeri, their applications, and detection methods. Background Art

[0002] Neoseiulus barkeri belongs to Arachnida, Acari, Parasitiformes, Phytoseiidea, Phytoseiidae, and can prey on a variety of small sap-sucking pests (mites), making it one of the important commercialized natural enemy groups. Neoseiulus barkeri has currently achieved industrial production and application. In recent years, with the continuous climate warming, the frequency and intensity of extreme climate events such as high temperature have increased significantly, and it is expected to continue to increase in the next few decades. High temperature will severely restrict the control effect of Neoseiulus barkeri.

[0003] By artificial screening, a high-temperature tolerant strain of Neoseiulus barkeri has been obtained, with significantly enhanced high-temperature tolerance and significantly improved control ability against major pests such as Tetranychus urticae under high temperature. Currently, there is no report on the correlation between the high-temperature tolerance ability of Neoseiulus barkeri and the molecular markers described in the present invention in this field.

[0004] Therefore, the present invention provides SNP molecular markers for high-temperature tolerance of Neoseiulus barkeri, their applications, and detection methods, which are used to screen high-temperature tolerant strains of Neoseiulus barkeri. Summary of the Invention

[0005] The object of the present invention is to provide SNP molecular markers for high-temperature tolerance of Neoseiulus barkeri, their applications, and detection methods, which are used to fill the blank in the classification field of normal-temperature strains and high-temperature tolerant strains of Neoseiulus barkeri and facilitate the screening of high-temperature tolerant strains of Neoseiulus barkeri.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a SNP molecular marker for high-temperature tolerance of Neoseiulus barkeri, and the SNP molecular marker is located at at least one of the following sites on chromosome 2 of the Neoseiulus barkeri genome:

[0008] (1) The 27,121,501bp site, where there is a G / A base mutation;

[0009] (2) The 27,174,672bp site, where there is a G / T base mutation;

[0010] (3) The 27,250,404bp site, where there is a C / T base mutation;

[0011] (4) At the 27,184,864 bp locus, there is a T / C base mutation.

[0012] Furthermore, the nucleotide sequences of the G / A base mutation at the 27,121,501 bp locus are shown as SEQ ID NO.1a / SEQ ID NO.1b;

[0013] The nucleotide sequences of the G / T base mutation at the 27,174,672 bp locus are shown as SEQ ID NO.2a / SEQ ID NO.2b;

[0014] The nucleotide sequences of the C / T base mutation at the 27,250,404 bp locus are shown as SEQ ID NO.3a / SEQ ID NO.3b;

[0015] The nucleotide sequences of the T / C base mutation at the 27,184,864 bp locus are shown as SEQ ID NO.4a / SEQ ID NO.4b.

[0016] The present invention provides the application of the SNP molecular marker in the non-diagnostic classification of the normal-temperature strain and the high-temperature resistant strain of Neoseiulus barkeri.

[0017] Furthermore, the application method is as follows:

[0018] If the base at the 27,121,501 bp locus on chromosome 2 of the genome of an individual of the normal-temperature strain of Neoseiulus barkeri is G, and / or the base at the 27,174,672 bp locus is G, and / or the base at the 27,250,404 bp locus is C, and / or the base at the 27,184,864 bp locus is T, then it is the normal-temperature strain;

[0019] If the base at the 27,121,501 bp locus on chromosome 2 of the genome of an individual of the normal-temperature strain of Neoseiulus barkeri is A, and / or the base at the 27,174,672 bp locus is T, and / or the base at the 27,250,404 bp locus is T, and / or the base at the 27,184,864 bp locus is C, then it is the high-temperature resistant strain.

[0020] The present invention provides a primer set for the SNP molecular marker. The primer set for the 27,121,501 bp locus is as follows:

[0021] Forward primer 1F: 5'-CTGCTAAACCAGCCAAAT-3';

[0022] Reverse primer 1R: 5'-GCTGAGCTTTCAGCGAGAC-3';

[0023] The primer set for the 27,174,672 bp locus is as follows:

[0024] Upstream primer 2F: 5′-AGCTATCATCCTCCCAGTT-3′;

[0025] Downstream primer 2R: 5′-GTTTCCTCGTCATAAATACATC-3′;

[0026] The primer sets for sites 27, 250, and 404 are:

[0027] Upstream primer 3F: 5′-CCGAATGGCAGGATGAGA-3′;

[0028] Downstream primer 3R: 5′-TTCCGAAGTGAGGAGATTGC-3′;

[0029] The primer set for position 27,184,864 is:

[0030] Upstream primer 4F: 5′-TCGCTGCTGGTGATGTTC-3′;

[0031] Downstream primer 4R: 5′-CGTCCGTTTCTTTCCTACTCT-3′.

[0032] The present invention provides a non-diagnostic detection method for the high temperature resistance of Neoseiulus barkeri, comprising the following steps:

[0033] Step 1: extracting the total DNA of the tested Neoseiulus barkeri;

[0034] Step: 2: Perform PCR amplification using the primer set as described above to obtain a DNA fragment containing the SNP site;

[0035] Step 3: Sequence the amplified product to detect the 27,121,501bp site, and / or the 27,174,672bp site, and / or the 27,250,404bp site, and / or the 27,184,864bp site, and determine whether the strain is a normal temperature strain or a high temperature resistant strain based on the base type.

[0036] Furthermore, the reaction system of the PCR amplification is: 12.5 μL of PrimeSTAR high-fidelity enzyme, 1 μL of each forward and reverse primer, 1 μL of DNA template, and 9.5 μL of ddH2O.

[0037] Furthermore, the PCR amplification program is: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; and finally post-extension at 72°C for 10 min.

[0038] Further, determine whether the strain is a normal-temperature strain or a high-temperature tolerant strain according to the base type, and the method is as follows:

[0039] If the base at the 27,121,501bp locus on chromosome 2 of the genome of an individual of the normal-temperature strain of Neoseiulus barkeri is G, and / or the base at the 27,174,672bp locus is G, and / or the base at the 27,250,404bp locus is C, and / or the base at the 27,184,864bp locus is T, then it is a normal-temperature strain;

[0040] If the base at the 27,121,501bp locus on chromosome 2 of the genome of an individual of the normal-temperature strain of Neoseiulus barkeri is A, and / or the base at the 27,174,672bp locus is T, and / or the base at the 27,250,404bp locus is T, and / or the base at the 27,184,864bp locus is C, then it is a high-temperature tolerant strain.

[0041] The present invention has at least the following beneficial effects:

[0042] The SNP molecular markers related to high-temperature tolerance of Neoseiulus barkeri provided by the present invention have a significant correlation with the normal-temperature strain and the high-temperature tolerant strain. By detecting the SNP molecular marker sites, it is possible to quickly identify the normal-temperature strain and the high-temperature tolerant strain of Neoseiulus barkeri, filling the blank in the classification field of the normal-temperature strain and the high-temperature tolerant strain of Neoseiulus barkeri, and having potential application value for large-scale molecular breeding of Neoseiulus barkeri. Specific embodiments

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following specific embodiments are used to further elaborate on the present invention. It should be understood that the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0044] The main objective of the present invention is to provide SNP molecular markers, applications and detection methods for the high-temperature tolerance of Neoseiulus barkeri, specifically as follows:

[0045] Example 1, this example is used to provide a SNP molecular marker for the high-temperature tolerance of Neoseiulus barkeri.

[0046] The SNP molecular marker is located at the 27,121,501 and / or 27,174,672 and / or 27,250,404 and / or 27,184,864bp loci on chromosome 2 of the Neoseiulus barkeri genome, and the SNP molecular marker has base mutations of G / A, G / T, C / T and T / C respectively.

[0047] Specifically, as shown in the following table:

[0048] Table 1 SNP molecular marker loci of Neoseiulus barkeri at normal temperature strain and high temperature tolerant strain

[0049]

[0050] The nucleotide sequences of the SNP molecular markers are shown in SEQ ID NO.1a (normal temperature strain), SEQ ID NO.1b (high temperature tolerant strain), SEQ ID NO.2a (normal temperature strain), SEQ ID NO.2b (high temperature tolerant strain), SEQ ID NO.3a (normal temperature strain), SEQ ID NO.3b (high temperature tolerant strain), SEQ ID NO.4a (normal temperature strain), and SEQ ID NO.4b (high temperature tolerant strain). The mutation positions are in italics and underlined as follows:

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] Example 2. This example is used to provide the specific forward and reverse primers of the SNP molecular marker for the high temperature tolerance of Neoseiulus barkeri in Example 1 above. The primer sequences are as follows:

[0057] Forward primer 1F: 5’-CTGCTAAACCAGCCAAAT-3’ (SEQ ID NO.1)

[0058] Reverse primer 1R: 5’-GCTGAGCTTTCAGCGAGAC-3’ (SEQ ID NO.1)

[0059] Forward primer 2F: 5’-AGCTATCATCCTCCCAGTT-3’ (SEQ ID NO.2)

[0060] Reverse primer 2R: 5’-GTTTCCTCGTCATAAATACATC-3’ (SEQ ID NO.2)

[0061] Forward primer 3F: 5’-CCGAATGGCAGGATGAGA-3’ (SEQ ID NO.3)

[0062] Reverse primer 3R: 5’-TTCCGAAGTGAGGAGATTGC-3’ (SEQ ID NO.3)

[0063] Upstream primer 4F: 5’-TCGCTGCTGGTGATGTTC-3’ (SEQ ID NO.4)

[0064] Downstream primer 4R: 5’-CGTCCGTTTCTTTCCTACTCT-3’ (SEQ ID NO.4)

[0065] Example 3. This example is used to provide a detection method for SNP molecular markers related to the high-temperature tolerance ability of *Neoseiulus barkeri*. The steps are as follows:

[0066] First, extract the total DNA of a single *Neoseiulus barkeri* to be tested. Using the total DNA as a template, perform PCR amplification with the specific forward and reverse primers in Example 2 above to obtain a gene fragment containing the SNP molecular marker. Compare the obtained gene fragment with the nucleotide sequence of the SNP molecular marker in Example 1, and then it can be determined whether this strain is a normal-temperature strain or a high-temperature-tolerant strain.

[0067] The PCR system is as follows: 12.5 μL of PrimeSTAR high-fidelity enzyme, 1 μL of each of the forward and reverse primers, 1 μL of DNA template, and 9.5 μL of ddH2O.

[0068] The PCR amplification program is as follows: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, for 35 cycles; finally, post-extension at 72°C for 10 min.

[0069] Example 4. This example mainly provides a specific example for the detection of SNP molecular markers of the normal-temperature strain and high-temperature-tolerant strain of *Neoseiulus barkeri*.

[0070] 1. Materials

[0071] 1.1 Test mite sources

[0072] Normal-temperature strain: *Neoseiulus barkeri* was introduced from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (in 2010), fed with *Aleuroglyphus ovatus*, and long-term large-scale reared in an artificial climate chamber (temperature: 25 ± 1°C; relative humidity: 70% - 80%; photoperiod: 14L:10D).

[0073] High-temperature-tolerant strain: Using the normal-temperature strain of *Neoseiulus barkeri* as the original material, through long-term high-temperature acclimation (35°C) and high-temperature training (45°C) screening, a high-temperature-tolerant strain with significantly improved high-temperature tolerance ability was obtained. Subsequently, high-temperature training was carried out every once in a while according to its population density to ensure that the resistance would not decline.

[0074] 1.2 Reagents

[0075] DNA extraction kit (Tiangen Biotech Co., Ltd., China), PrimeSTAR (TaKaRa Bio Inc., Japan).

[0076] 2. Methods

[0077] 2.1 Total DNA extraction of the normal-temperature strain and high-temperature resistant strain of Neoseiulus barkeri

[0078] Total DNA was extracted from the normal-temperature strain and high-temperature resistant strain of Neoseiulus barkeri using a DNA extraction kit respectively.

[0079] 2.2 SNP molecular marker analysis of the normal-temperature strain and high-temperature resistant strain of Neoseiulus barkeri

[0080] Based on the whole-genome resequencing of the normal-temperature strain and high-temperature resistant strain of Neoseiulus barkeri, 4 SNP molecular markers were found by manual search. The SNP molecular markers are located at the 27,121,501, 27,174,672, 27,250,404, and 27,184,864 bp sites on chromosome 2 of the Neoseiulus barkeri genome, and the SNP molecular markers have base mutations of G / A, G / T, C / T, and T / C respectively.

[0081] 2.3 Nucleotide sequence preference of the Neoseiulus barkeri containing the SNP molecular marker

[0082] The nucleotide sequences containing the SNP molecular markers are preferably as shown in SEQ ID NO.1a, SEQ ID NO.1b, SEQ ID NO.2a, SEQ ID NO.2b, SEQ ID NO.3a, SEQ ID NO.3b, SEQ ID NO.4a, and SEQ ID NO.4b in the above embodiments.

[0083] 2.4 Primer design for the nucleotide sequence of Neoseiulus barkeri

[0084] A primer set for detecting the SNP molecular marker described in the above technical solution is provided. The primer set includes an upstream primer and a downstream primer, specifically as follows:

[0085] Upstream primer 1F: 5’-CTGCTAAACCAGCCAAAT-3’ (SEQ ID NO.1)

[0086] Downstream primer 1R: 5’-GCTGAGCTTTCAGCGAGAC-3’ (SEQ ID NO.1)

[0087] Upstream primer 2F: 5’-AGCTATCATCCTCCCAGTT-3’ (SEQ ID NO.2)

[0088] Downstream primer 2R: 5’-GTTTCCTCGTCATAAATACATC-3’ (SEQ ID NO.2)

[0089] Upstream primer 3F: 5’-CCGAATGGCAGGATGAGA-3’ (SEQ ID NO.3)

[0090] Downstream primer 3R: 5’-TTCCGAAGTGAGGAGATTGC-3’ (SEQ IDNO.3)

[0091] Upstream primer 4F: 5’-TCGCTGCTGGTGATGTTC-3’ (SEQ ID NO.4)

[0092] Downstream primer 4R: 5’-CGTCCGTTTCTTTCCTACTCT-3’ (SEQ ID NO.4)

[0093] The above primers were designed to perform PCR amplification on the normal-temperature strain and high-temperature resistant strain of *Neoseiulus barkeri*. The reaction system was as follows: 12.5 μL of PrimeSTAR high-fidelity enzyme, 1 μL of each forward and reverse primer, 1 μL of DNA template, and 9.5 μL of ddH2O. The PCR amplification program was: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, for 35 cycles; finally, post-extension at 72°C for 10 min. The PCR products were sent to BGI for sequencing.

[0094] By comparing the sequencing results, it was found that the SNP molecular marker had a significant correlation with the high-temperature resistance ability of *Neoseiulus barkeri*. The SNP sites at positions 27,121,501, 27,174,672, 27,250,404, and 27,184,864 bp on chromosome 2 of the genome of individuals of the normal-temperature strain of *Neoseiulus barkeri* were G, G, C, and T respectively, while those of individuals of the high-temperature resistant strain were A, T, T, and C respectively.

[0095] From the above examples, it can be concluded that the SNP molecular marker related to high-temperature resistance of *Neoseiulus barkeri* provided by the present invention has a significant correlation with the normal-temperature strain and the high-temperature resistant strain. By detecting the SNP molecular marker sites, it is possible to quickly identify the normal-temperature strain and the high-temperature resistant strain of *Neoseiulus barkeri*, which has potential application value for large-scale molecular breeding of *Neoseiulus barkeri*.

[0096] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. A SNP molecular marker for the high-temperature tolerance of Neoseiulus barkeri, characterized in that, The SNP molecular marker is located at at least one of the following sites on chromosome 2 of the genome of *Neoseiulus barkeri*: (1) The site at 27,121,501 bp, with a G / A base mutation; (2) The site at 27,174,672 bp, with a G / T base mutation; (3) The site at 27,250,404 bp, with a C / T base mutation; (4) The site at 27,184,864 bp, with a T / C base mutation.

2. The SNP molecular marker for high-temperature tolerance of Neoseiulus barkeri according to claim 1, characterized in that, The nucleotide sequence of the G / A base mutation at the 27,121,501 bp site is shown as SEQ ID NO.1a / SEQ ID NO.1b; The nucleotide sequence of the G / T base mutation at the 27,174,672 bp site is shown as SEQ ID NO.2a / SEQ ID NO.2b; The nucleotide sequence of the C / T base mutation at the 27,250,404 bp site is shown as SEQ ID NO.3a / SEQ ID NO.3b; The nucleotide sequence of the T / C base mutation at the 27,184,864 bp site is shown as SEQ ID NO.4a / SEQ ID NO.4b.

3. The application of the SNP molecular marker according to claim 1 in the non-diagnostic classification of the normal-temperature strain and the high-temperature-tolerant strain of *Neoseiulus barkeri*.

4. The application according to claim 3, characterized in that, The application method is as follows: If the site at 27,121,501 bp on chromosome 2 of the genome of an individual of the normal-temperature strain of *Neoseiulus barkeri* is G, and / or the site at 27,174,672 bp is G, and / or the site at 27,250,404 bp is C, and / or the site at 27,184,864 bp is T, then it is the normal-temperature strain; If the site at 27,121,501 bp on chromosome 2 of the genome of an individual of the normal-temperature strain of *Neoseiulus barkeri* is A, and / or the site at 27,174,672 bp is T, and / or the site at 27,250,404 bp is T, and / or the site at 27,184,864 bp is C, then it is the high-temperature-tolerant strain.

5. A primer set for detecting the SNP molecular marker as described in claim 1, characterized in that, The primer set for the 27,121,501 bp site is: Forward primer 1F: 5'-CTGCTAAACCAGCCAAAT-3'; Reverse primer 1R: 5'-GCTGAGCTTTCAGCGAGAC-3'; The primer set for the 27,174,672 bp site is: Forward primer 2F: 5'-AGCTATCATCCTCCCAGTT-3'; Reverse primer 2R: 5'-GTTTCCTCGTCATAAATACATC-3'; The primer set for the 27,250,404 site is: Forward primer 3F: 5'-CCGAATGGCAGGATGAGA-3'; Reverse primer 3R: 5'-TTCCGAAGTGAGGAGATTGC-3'; The primer set for the 27,184,864 site is: Forward primer 4F: 5'-TCGCTGCTGGTGATGTTC-3'; Downstream primer 4R: 5'-CGTCCGTTTCTTTCCTACTCT-3'.

6. A non-diagnostic detection method for the high temperature tolerance of Neoseiulus barkeri, characterized in that, It includes the following steps: Step 1: Extract the total DNA of the to-be-detected Neoseiulus barkeri; Step 2: Perform PCR amplification using the primer set as described in claim 5 to obtain a DNA fragment containing SNP sites; Step 3: Sequence the amplification product to detect the 27,121,501bp site, and / or the 27,174,672bp site, and / or the 27,250,404bp site, and / or the 27,184,864bp site, and determine whether the strain is a normal-temperature strain or a high-temperature-resistant strain according to the base type.

7. A non-diagnostic detection method for the high-temperature tolerance of Neoseiulus barkeri according to claim 6, characterized in that The reaction system for the PCR amplification is as follows: 12.5 μL of PrimeSTAR high-fidelity enzyme, 1 μL of each of the forward and reverse primers, 1 μL of DNA template, and 9.5 μL of ddH2O.

8. A non-diagnostic detection method for the high temperature tolerance of Neoseiulus barkeri according to claim 6, characterized in that The PCR amplification program is as follows: Pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, for 35 cycles; finally post-extension at 72°C for 10 min.

9. The non-diagnostic detection method for the high temperature tolerance of Neoseiulus barkeri according to claim 6, characterized in that, Determine whether the strain is a normal-temperature strain or a high-temperature-resistant strain according to the base type, and the method is as follows: If the 27,121,501bp site on chromosome 2 of the genome of an individual of the normal-temperature strain of Neoseiulus barkeri is G, and / or the 27,174,672bp site is G, and / or the 27,250,404bp site is C, and / or the 27,184,864bp site is T, then it is a normal-temperature strain; If the 27,121,501bp site on chromosome 2 of the genome of an individual of the normal-temperature strain of Neoseiulus barkeri is A, and / or the 27,174,672bp site is T, and / or the 27,250,404bp site is T, and / or the 27,184,864bp site is C, then it is a high-temperature-resistant strain.

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