Molecular marker for identifying gender of giant spiny frog and identification method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a molecular marker for identifying the gender of giant spiny frogs and an identification method. The invention provides two groups of molecular markers for accurately identifying the gender of the giant spiny frog for the first time, both the two groups of molecular markers can accurately and effectively identify the gender of the giant spiny frog, and the blank of molecular marker gender identification of the giant spiny frog is filled.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a molecular marker for identifying the sex of Quasipaa spinosa and an identification method therefor. Background Art

[0002] Quasipaa spinosa belongs to Amphibia, Anura, Dicroglossidae, and Quasipaa, and is distributed in southern provinces of China such as Zhejiang, Anhui, Jiangxi, and Fujian, as well as Vietnam and other places. Quasipaa spinosa has important ecological value and scientific research value. At the same time, due to its tender and white meat, it also has important economic value.

[0003] At present, little is known about the sex determination mechanism of Quasipaa spinosa. Small spines (thorns) grow on the chest and abdomen of adult male Quasipaa spinosa, which can be used to determine sex. However, these small spines will fall off during the non-breeding period, which easily leads to misidentification. Moreover, at the tadpole and juvenile stages of Quasipaa spinosa, small spines have not grown on the chest and abdomen of males, and the external morphological differences are not obvious. Therefore, at present, except for dissection, there is no accurate sex identification method, and there is a lack of a method for accurately identifying sex without harming young frogs or tadpoles.

[0004] It is difficult to accurately distinguish and identify the physiological sex and genetic sex of live Quasipaa spinosa by relying on traditional methods, which hinders the artificial breeding of Quasipaa spinosa. Although specific molecular markers are reliable sex identification means, no molecular markers that can accurately and effectively identify the sex of Quasipaa spinosa have yet appeared.

[0005] Therefore, there is an urgent need to develop a method for accurately and conveniently identifying the sex of Quasipaa spinosa. Summary of the Invention

[0006] The purpose of the present invention is to provide a molecular marker for identifying the sex of Quasipaa spinosa and an identification method therefor.

[0007] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is: a set of primer pairs, the primer pairs include: the upstream primer and the downstream primer of primer pair 457019; the upstream primer sequence of primer pair 457019 is shown as SEQ ID NO:1, and the downstream primer sequence is shown as SEQ ID NO:2; and / or; the upstream primer and the downstream primer of primer pair 316336; the upstream primer sequence of primer pair 316336 is shown as SEQ ID NO:3, and the downstream primer sequence is shown as SEQ ID NO:4; and / or; the upstream primer and the downstream primer of primer pair 696744; the upstream primer sequence of primer pair 696744 is shown as SEQ ID NO:5, and the downstream primer sequence is shown as SEQ ID NO:6.

[0008] Correspondingly, a set of primer groups, the primer groups include the upstream primer and the downstream primer of primer pair 1139149, the upstream primer sequence is as shown in SEQ ID NO:7, and the downstream primer sequence is as shown in SEQ ID NO:8.

[0009] Correspondingly, the application of the primer group in identifying the sex of Rana spinosa.

[0010] Correspondingly, a method for identifying the sex of Rana spinosa, extracting the genomic DNA of Rana spinosa to be identified for sex, using the primer pair to perform PCR amplification on the genomic DNA of Rana spinosa, and identifying the sex of Rana spinosa based on the amplification product. If a specific band appears near 250bp in the amplification product of primer pair 316336, it is identified as male, and if no specific band appears, it is identified as female; and / or; if a specific band appears near 100bp in the amplification product of primer pair 696744, it is male, and if no specific band appears, Rana spinosa is identified as female; and / or; if a specific band appears near 250bp in the amplification product of primer pair 457019, Rana spinosa is identified as male; if no specific band appears, Rana spinosa is identified as female. "Near" means ±10bp at this position, for example, "near 250bp" means 250±10bp.

[0011] Correspondingly, a method for identifying the sex of Rana spinosa, extracting the genomic DNA of Rana spinosa to be identified for sex, using the primer pair to perform PCR amplification on the genomic DNA of Rana spinosa, and identifying the sex of Rana spinosa based on the amplification product.

[0012] Correspondingly, a method for identifying the sex of Rana spinosa, the method uses the sequence locus at position 139661202 of chromosome 1 of Rana spinosa to judge. If the sequenced base at this locus is homozygous, it is identified as female, and if the base is heterozygous, it is identified as male.

[0013] Correspondingly, a kit, reagent, and test paper containing the primer group.

[0014] The present invention has the following beneficial effects: The present invention for the first time provides two sets of molecular markers that can be used to accurately identify the sex of Rana spinosa, both of which can accurately and effectively identify the sex of Rana spinosa, filling the blank of molecular marker sex identification of Rana spinosa. Description of the Drawings

[0015] Figure 1 It is the gel electrophoresis diagram of the amplification product of Rana spinosa DNA amplified by primer pair 696744;

[0016] Figure 2 It is the gel electrophoresis diagram of the amplification product of Rana spinosa DNA amplified by primer pair 316336;

[0017] Figure 3Gel electrophoresis pattern of the amplification product of Paa spinosa DNA amplified using primer pair 457019;

[0018] Figure 4 Peak pattern of the amplification product of Paa spinosa DNA amplified using primer pair 1139149. Detailed implementation manners

[0019] The present invention provides two primer sets capable of effectively identifying the sex of Paa spinosa, and the sequences of the primer sets are shown in Table 1.

[0020] Table 1 Sequence comparison table of primer sets 1 and 2

[0021]

[0022] Based on the primer sets, the present invention also correspondingly provides a method for identifying the sex of Paa spinosa.

[0023] Method 1: Using primer set 1; the specific steps include: extracting the genomic DNA of the Paa spinosa to be tested, and performing PCR amplification using the primer pairs of primer set 1.

[0024] If a specific band appears near 250bp in the amplification product of the 316336 primer pair, it is identified as male; if no specific band appears, it is identified as female.

[0025] If a specific band appears near 100bp in the amplification product of the 696744 primer pair, it is male; if no specific band appears, the Paa spinosa is identified as female.

[0026] If a specific band appears near 250bp in the amplification product of the 457019 primer pair, the Paa spinosa is identified as male; if no specific band appears, the Paa spinosa is identified as female.

[0027] The 3 primer pairs in primer set 1 can be used alone or in combination for sex identification. Through tests by the inventor's research group, these 3 primer pairs are highly correlated with the sex of Paa spinosa, and no counterexample of "bands appear in males and no bands appear in females" has been found in actual measurements.

[0028] If the three primer pairs in primer set 1 are used for identification at the same time, and bands appear at the corresponding positions in the amplification products of all three primer pairs, or bands appear at the corresponding positions in the amplification products of two primer pairs, it is identified as male; otherwise, it is identified as female.

[0029] If any two pairs of primers in Primer Set 1 are used for identification simultaneously, bands will appear at the corresponding positions in the amplification products of both pairs of primers, and it is identified as male; if no bands appear, it is identified as female. If there is a situation where the amplification product of one pair of primers has a band and the amplification product of the other pair of primers has no band, a repeated test is carried out, or a third pair of primers is introduced for amplification until bands appear at the corresponding positions in the amplification products of at least two pairs of primers (identified as male), or no bands appear (identified as female).

[0030] Method 2: It is carried out using Primer Set 2. The specific steps include: extracting the genomic DNA of the to-be-detected spiny frog, and performing PCR amplification using the primer pairs of Primer Set 2.

[0031] Sequence the obtained PCR amplification product to obtain a peak map. If it shows heterozygous double peaks at the sex-linked SNP locus (position 139661202 on chromosome 1), it is identified as male; if it shows homozygous single peaks at this locus, it is identified as female.

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The obtained data are all the averages obtained after at least 3 repetitions, and all repetitions obtain valid data.

[0033] Example 1: Identifying the sex of spiny frogs using Primer Set 1

[0034] Collect 10 male spiny frogs and 10 female spiny frogs from Jiangxi Province. The method for judging the sex of spiny frogs is: observing and confirming through gonads (dissecting and observing the gonadal tissues of individuals, if it is an ovary, it is female, if it is a testis, it is male), appearance, etc.

[0035] 1. Sacrifice the spiny frog, and extract DNA from the muscle tissue using an animal tissue DNA extraction kit as the sample DNA.

[0036] 2. Perform PCR amplification

[0037] Configure a 25 μL PCR system: 2×master, 2×masterMIX (12.5 μL), the upstream and downstream primers of each primer pair in Table 1 of Primer Set 1 (1.5 μL each), sample DNA (1 μL), and sterilized double-distilled water (8.5 μL). In Primer Set 1, each primer pair is configured according to the said PCR system and used to amplify the sample DNA respectively.

[0038] PCR procedure: Pre-denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 30 seconds, with a final extension at 72°C for 5 minutes, and storage at 4°C.

[0039] 3. Gel electrophoresis: After the PCR procedure, perform electrophoresis using a 1.5% agarose gel at 100V for 30 minutes, and the detection results are as Figure 1 , Figure 2 and Figure 3 shown.

[0040] As Figures 1 to 3 shown: Among the amplification products obtained by amplifying the DNA of Rana spinosa samples using the 696744 primer pair, specific bands all appeared near 107bp for the male samples' corresponding amplification products, and no specific bands appeared at this position for the female samples' corresponding amplification products. Among the amplification products obtained by amplifying the DNA of Rana spinosa samples using the 316336 primer pair, specific bands all appeared near 251bp for the male samples' corresponding amplification products, and no specific bands appeared at this position for the female samples' corresponding amplification products. Among the amplification products obtained by amplifying the DNA of Rana spinosa samples using the 457019 primer pair, specific bands all appeared near 246bp for the male samples' corresponding amplification products, and no specific bands appeared at this position for the female samples' corresponding amplification products.

[0041] The results of identifying the genders of 20 Rana spinosa using this method were completely consistent with those identified by gonad and appearance observation, proving that the primer pairs and method provided by the present invention are universal, and the identification results are accurate and effective.

[0042] Example 2: Identifying the gender of Rana spinosa using primer set 2

[0043] Collect a total of 27 Rana spinosa from Tongshan County, Hubei Province; Yizhang County, Hunan Province; Yihuang County, Jiangxi Province; Suichuan County, Jiangxi Province; Tiantai County, Zhejiang Province; Jianyang District, Fujian Province; and Dehua County, Fujian Province, including 16 males and 11 females. The gender of Rana spinosa was determined by confirming through gonad dissection and appearance observation.

[0044] Extract the DNA of Rana spinosa according to the method of Example 1, and use the primer pairs in primer set 2 to amplify the sample DNA according to the same PCR system and PCR procedure as in Example 1. After the PCR procedure, send the product to a sequencing company for sequencing. The results are as Figure 4 shown.

[0045] The results showed that: at the sex-linked SNP locus (position 139661202 on chromosome 1 of the genome), it showed a heterozygous double peak (manifested as degenerate bases, C / G heterozygous, labeled as S), and was identified as male; at this locus, it showed a homozygous single peak and was identified as female. The results of identifying 27 spiny-breasted frogs using this method were completely consistent with the results of gonad, appearance and anatomical identification, proving that the position 139661202 on chromosome 1 of the genome is indeed highly correlated with the sex of spiny-breasted frogs, and the primer pairs and methods provided by the present invention have universality, and the identification results are accurate and effective.

[0046] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer set, characterized in that: The primer set includes: an upstream primer and a downstream primer of primer pair 457019; the upstream primer sequence of primer pair 457019 is shown in SEQ ID NO:1, and the downstream primer sequence is shown in SEQ ID NO:2; and / or; an upstream primer and a downstream primer of primer pair 316336; the upstream primer sequence of primer pair 316336 is shown in SEQ ID NO:3, and the downstream primer sequence is shown in SEQ ID NO:4; and / or; an upstream primer and a downstream primer of primer pair 696744; the upstream primer sequence of primer pair 696744 is shown in SEQ ID NO:5, and the downstream primer sequence is shown in SEQ ID NO:

6.

2. A primer set, characterized in that: The primer set includes an upstream primer and a downstream primer of primer pair 1139149, the upstream primer sequence is shown in SEQ ID NO:7, and the downstream primer sequence is shown in SEQ ID NO:

8.

3. Use of the primer set according to claim 1 in identifying the sex of Rana spinosa.

4. Use of the primer set according to claim 2 in identifying the sex of Rana spinosa.

5. A method for identifying the sex of Rana spinosa, characterized in that: Extract the genomic DNA of the spiny-chested frog whose sex is to be identified, perform PCR amplification on the genomic DNA of the spiny-chested frog using the primer pair of claim 1, and identify the sex of the spiny-chested frog based on the amplified product.

6. The method according to claim 5, characterized in that: If the amplification product of primer pair 316336 shows a specific band near 250 bp, it is identified as male, and if no specific band appears, it is identified as female; and / or; If the amplification product of primer pair 696744 shows a specific band near 100 bp, it is male, and if no specific band appears, the spiny-chested frog is identified as female; and / or; If a specific band appears near 250 bp in the amplification product of primer pair 457019, the spiny-chested frog is identified as a male; if no specific band appears, the spiny-chested frog is identified as a female.

7. A method for identifying the sex of Rana spinosa, characterized in that: Extract the genomic DNA of the spiny-chested frog whose sex is to be identified, perform PCR amplification on the genomic DNA of the spiny-chested frog using the primer pair of claim 2, and identify the sex of the spiny-chested frog based on the amplified product.

8. A method for identifying the sex of Rana spinosa, characterized in that: The method uses the sequence site 139661202 of chromosome 1 of Rana spinosa to make judgments. If the sequenced base of the site is homozygous, it is identified as female, and if the base is heterozygous, it is identified as male.

9. A kit, reagent, or test paper comprising the primer set according to claim 1.

10. A kit, reagent, or test paper comprising the primer set according to claim 2.

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