A specific primer for female Shinisaurus crocodilurus and a method for sex identification

Abstract

The present invention discloses a female-specific primer for Shinisaurus crocodilurus and a method for sex identification. Genomic DNA of the Shinisaurus crocodilurus to be tested is extracted, and then amplified with the above-mentioned specific primer. After detection by agarose gel electrophoresis, if there is a specific band at 557 bp, it indicates that the sample corresponds to a female Shinisaurus crocodilurus; if there is no band, it is a male Shinisaurus crocodilurus. Since Shinisaurus crocodilurus has no obvious secondary sexual characteristics, there is a lack of a reliable method for identifying the sex of Shinisaurus crocodilurus in the prior art, especially for juvenile Shinisaurus crocodilurus, it is impossible to determine the sex. The method adopted in the present invention is a molecular biology method based on genomic information, and is detected by PCR method, which has the advantages of high repeatability, strong reliability, high throughput, and independence from the experience of breeders. Whether it is an adult or a juvenile Shinisaurus crocodilurus, its sex can be accurately determined.

Description

Technical Field

[0001] The present invention belongs to the field of sex identification, and particularly relates to a female-specific primer for Shinisaurus crocodilurus and a method for sex identification thereof. Background Art

[0002] Shinisaurus crocodilurus is a first-class protected wild animal in China. It belongs to the class Reptilia, order Squamata, family Shinisauridae, and genus Shinisaurus, and is known as the "living fossil" of reptiles. At present, releasing artificially bred Shinisaurus crocodilurus into the wild is an important measure for its protection. When deeply studying the reproductive biology of Shinisaurus crocodilurus, it is necessary to accurately identify the male and female individuals of Shinisaurus crocodilurus first, and it is also necessary to identify the male and female individuals as early as possible during the artificial breeding process, so as to reasonably match the male-female ratio.

[0003] At present, little is known about the sex determination mechanism of Shinisaurus crocodilurus. The chromosome karyotype of Shinisaurus crocodilurus shows that there are no heteromorphic chromosomes. The secondary sexual characteristics of Shinisaurus crocodilurus are not obvious. For adult Shinisaurus crocodilurus, there are sexual dimorphisms in body color and local characteristics such as the head and trunk in statistical data. For example, the head, chest and abdomen of adult male Shinisaurus crocodilurus are light blue or bright red, while those of females are light yellow or light red. In fact, the body color variation of Shinisaurus crocodilurus is richer than previously reported, and the color pattern types of the same individual can also change with age. In the sex identification of individuals, relying on appearance to distinguish the sex of Shinisaurus crocodilurus has low reliability and depends on the long-term observation experience of breeders. For juvenile Shinisaurus crocodilurus, the male and female have the same appearance, and it is impossible to identify the sex by observing their external morphology. Therefore, an accurate and convenient sex identification method is needed in this field. Summary of the Invention

[0004] Aiming at the current lack of a reliable and convenient method for identifying the sex of Shinisaurus crocodilurus, the purpose of the present invention is to provide an accurate, repeatable and convenient method for sex identification of Shinisaurus crocodilurus. By non-invasively collecting the DNA of saliva or dandruff, and applying male and female specific primers, the sex of Shinisaurus crocodilurus can be quickly determined under the conditions of PCR and agarose gel electrophoresis, without relying on the breeding experience of Shinisaurus crocodilurus breeders.

[0005] The first object of the present invention is to provide a female-specific primer for Shinisaurus crocodilurus that can accurately determine the sex of both adult and juvenile Shinisaurus crocodilurus, specifically:

[0006] exW-F1: 5’-ATTCACTTCGAGGGAGAATGA-3’

[0007] exW-R1: 5’-GCATCGGTAATCGCATTGACA-3’.

[0008] The second object of the present invention is to provide a method for sex identification of Shinisaurus crocodilurus, comprising the following steps:

[0009] Extract the genomic DNA of the Chinese crocodile lizard to be tested, and then amplify it with the above specific primers. After detecting by agarose gel electrophoresis, if there is a specific band at 557 bp, it indicates that the sample corresponds to a female Chinese crocodile lizard; if there is no band, it is a male Chinese crocodile lizard.

[0010] Preferably, the reaction system is as follows: 12.5 μL of 2× Taq PCR Mix, 1.0 μL of DNA template, and the usage amount of the template can be adjusted according to the concentration of DNA. 0.5 μL of each of the forward and reverse primers with a concentration of 10 μmol / L, and make up to 25 μL with sterile deionized water.

[0011] Preferably, the PCR reaction procedure is as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 57 - 63 °C for 30 s, extension at 72 °C for 30 s, for 35 cycles; final extension at 72 °C for 8 min.

[0012] Since the Chinese crocodile lizard has no obvious secondary sexual characteristics, there is a lack of a reliable method for identifying male and female in the prior art, especially for juvenile Chinese crocodile lizards, it is impossible to determine the gender. The method adopted in the present invention is a molecular biology method based on genomic information, and is detected by the PCR method, which has the advantages of high repeatability, strong reliability, high throughput, and not relying on the experience of breeders. Whether it is an adult or a juvenile Chinese crocodile lizard, its gender can be accurately determined. Description of the Drawings

[0013] Figure 1 It is the identification of the gender of the Chinese crocodile lizard under the annealing condition of 57 °C; M1 - M8: samples of male Chinese crocodile lizards; F1 - F6: samples of female Chinese crocodile lizards; M: DL2000 DNA molecular weight standard, and the band sizes from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp in turn.

[0014] Figure 2 It is the optimization of the annealing temperature for amplification with the female - specific primer. T1 - T8: the annealing temperatures for PCR amplification are 55 °C, 55.7 °C, 56.9 °C, 58.8 °C, 61.1 °C, 63 °C, 64.3 °C, and 65 °C in turn; M: DL2000 DNA molecular weight standard, and the band sizes from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp in turn.

[0015] Figure 3 It is the identification of the gender of the Chinese crocodile lizard under the annealing condition of 60 °C; M1 - M8: samples of male Chinese crocodile lizards; F1 - F6: samples of female Chinese crocodile lizards; M: DL2000 DNA molecular weight standard, and the band sizes from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp in turn.

[0016] Figure 4 Sex identification of Chinese crocodile lizards under the annealing condition of 63°C. F1 - F6: Samples of female Chinese crocodile lizards; M1 - M8: Samples of male Chinese crocodile lizards; M: DL2000 DNA molecular weight standard. The band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively. Specific implementation mode

[0017] The following examples are further illustrations of the present invention, rather than limitations thereof.

[0018] Collect the pharyngeal swabs of Chinese crocodile lizards without damage using a sterile cotton swab. Since there are cells of Chinese crocodile lizards remaining on it, extract the total DNA using any nucleic acid extraction kit to obtain the genomic DNA of Chinese crocodile lizards. Then perform amplification using the above specific primers. After agarose gel electrophoresis detection, if there is a specific band at 557bp, it indicates that the sample corresponds to a female Chinese crocodile lizard; if there is no band, it is a male Chinese crocodile lizard.

[0019] The sequence of the amplification product is shown in SEQ ID NO.1. After comparison with the GenBank Nr database, it is found that no similar sequence is aligned to this segment, and no annotation information is obtained.

[0020] SEQ ID NO.1

[0021] ATTCACTTCGAGGGAGAATGATGCCAATGGCCTGCGCTTCCAAGCAGAGCGTGCCTTCTTCCGGATCTGCCTCCAGGCAGCCGCTTCTGCTCCGCCGCAAAGGAAAGGGGGAGAAAGGCTAGAGAAACAAATGCCCTGCCCAGATGACATCCTCCCGGTGGTACATCTCTCTTGTCTGGCATCCTTGCGTGATAACTATGGGACAGATTACTATGAGATAGAATCAAACGGCGGATCTGTTCCTGAGCATTACATCGATGCTGTGAACCGTAATGTCCTTTTATATTTAAATTCCCTCCATCTGCTTTATTGATTCCTCTCTGCTGCAGCGGAGCCAGAATCGGGGAAATACAAGGAAGGCCCGTCTGTATTATTCATTCTGGGGCATTATTATTAAAACCCGGCGGGCGCAAGGGAGGAGTGTTGGGGGGGGGGGTCTTAATATTACTCCAAAATGTTCCAACGAAGCTTCAGCTGGTCCCAATCAGGTCCCCGATAGATCCTCTAAATCCGGCATGATTGTTCCTTTTGAAATATGTCAATGCGATTACCGATGC

[0022] Example 1:

[0023] Taking 8 male crocodile lizards and 6 female crocodile lizards whose genders have been confirmed by pregnancy status, presence or absence of hemipenes, appearance, etc. under artificial breeding conditions as examples, the specificity of the primers provided by the present invention was verified.

[0024] The total DNA of the pharyngeal swabs of crocodile lizards was extracted using a nucleic acid kit.

[0025] A 25-μL reaction system was prepared: 12.5 μL of 2×Taq PCR Mix, 1.0 μL of DNA template, and the template usage can be adjusted according to the DNA concentration. 0.5 μL of each of the forward and reverse primers with a concentration of 10 μmol / L were added, and sterile deionized water was used to make up to 25 μL.

[0026] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, for 35 cycles; final extension at 72°C for 8 min. The annealing temperature can be selected within a certain range. For details, see Example 2.

[0027] After the PCR program is completed, electrophoresis is performed on a 1% agarose gel at a voltage of 100 V for 20 - 30 min, and the detection results are as Figure 1 shown.

[0028] The size of the target band amplified by the primers provided by the present invention is 557 bp. By comparing with the DNA molecular weight standard (lane M), it can be seen that there are no specific bands near 500 bp in male samples (lanes M1 - M8), while there is a specific band in each female sample (lanes F1 - F6). Thus, the sex of Chinese crocodile lizards can be identified.

[0029] Example 2:

[0030] Using the DNA of female sample F6 as a template, explore the annealing temperature range for amplification by the primers provided by the present invention.

[0031] Prepare a 25 - μL reaction system: 12.5 μL of 2×Taq PCR Mix, 1.0 μL of DNA template, and the template usage can be adjusted according to the DNA concentration. 0.5 μL of each forward and reverse primer with a concentration of 10 μmol / L, and make up to 25 μL with sterile deionized water.

[0032] The PCR reaction program is: pre - denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing temperatures are set to 8 temperatures in the range of 55 - 65°C, annealing time is 30 s, extension at 72°C for 30 s, cycle 35 times; final extension at 72°C for 8 min.

[0033] After the PCR program is completed, electrophoresis is performed on a 1% agarose gel at a voltage of 100 V for 20 - 30 min, and the detection results are as Figure 2 shown.

[0034] The size of the target band amplified by the primers provided by the present invention is 557 bp. By comparing with the DNA molecular weight standard (lane M), it can be seen that when the annealing temperature is set between 55 - 65°C, specific bands can be amplified from female samples near 500 bp. However, when the annealing temperature is higher than 63°C, the band brightness of the amplification product is lower (lanes T7 - T8). Thus, it can be known that when using the specific primers provided by the present invention for sex identification of Chinese crocodile lizards, the annealing temperature can be preferentially set to 63°C and below.

[0035] Example 3:

[0036] Taking 8 male Chinese crocodile lizards and 6 female Chinese crocodile lizards with confirmed genders under artificial breeding conditions as examples, verify the specificity of the primers provided by the present invention.

[0037] Extract the total DNA of the throat swabs of Chinese crocodile lizards using a nucleic acid kit.

[0038] Prepare a 25-μL reaction system: 12.5 μL of 2× Taq PCR Mix, 1.0 μL of DNA template. The amount of template used can be adjusted according to the DNA concentration. 0.5 μL each of forward and reverse primers with a concentration of 10 μmol / L, and make up to 25 μL with sterile deionized water.

[0039] The PCR reaction program is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for 35 cycles; final extension at 72°C for 5 min.

[0040] After the PCR program is completed, use 1% agarose gel and electrophorese at 100 V for 20 - 30 min. The detection results are as Figure 3 shown.

[0041] The size of the target band amplified by the primers provided by the present invention is 557 bp. By comparing with the DNA molecular weight standard (lane M), it can be seen that there are no specific bands in the male samples (lanes M1 - M8) near 500 bp, while there is a specific band in each of the female samples (lanes F1 - F6). Thus, the sex of the crocodile lizard can be identified.

[0042] Example 4:

[0043] Taking 8 male crocodile lizards and 6 female crocodile lizards with confirmed genders under artificial breeding conditions as examples respectively, verify the specificity of the primers provided by the present invention.

[0044] Extract the total DNA of the pharyngeal swabs of crocodile lizards using a nucleic acid kit.

[0045] Prepare a 25-μL reaction system: 12.5 μL of 2× Taq PCR Mix, 1.0 μL of DNA template. The amount of template used can be adjusted according to the DNA concentration. 0.5 μL each of forward and reverse primers with a concentration of 10 μmol / L, and make up to 25 μL with sterile deionized water.

[0046] The PCR reaction program is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s, for 35 cycles; final extension at 72°C for 8 min.

[0047] After the PCR program is completed, use 1% agarose gel and electrophorese at 100 V for 20 - 30 min. The detection results are as Figure 4 shown.

[0048] The size of the target band amplified by the primers provided by the present invention is 557 bp. By comparing with the DNA molecular weight standard (lane M), it can be seen that there are no specific bands in the male samples (lanes M1 - M8) near 500 bp, while there is a specific band in each of the female samples (lanes F1 - F6). Thus, the sex of the crocodile lizard can be identified.

Claims

1. A pair of sex-specific primers for Shinisaurus crocodilurus, characterized in that, Specifically: exW-F1: 5’-ATTCACTTCGAGGGAGAATGA-3’ exW-R1: 5’-GCATCGGTAATCGCATTGACA-3’.

2. A method for sex identification of Shinisaurus crocodilurus, characterized in that, It includes the following steps: Extract the genomic DNA of the Chinese crocodile lizard to be tested, then amplify it with the specific primers described in claim 1. After detecting by agarose gel electrophoresis, if there is a specific band at 557bp, it indicates that the Chinese crocodile lizard to be tested corresponds to a female Chinese crocodile lizard; if there is no band, it is a male Chinese crocodile lizard.

3. The identification method according to claim 2, wherein For the amplification mentioned above, the reaction system is: 2×TaqPCRMix 12.5μL, DNA template 1.0μL. The usage amount of the template is adjusted according to the DNA concentration. Each of the forward and reverse primers with a concentration of 10μmol / L is 0.5μL, and sterile deionized water is added to make up to 25μL.

4. The identification method according to claim 2, wherein, For the amplification mentioned above, the PCR reaction program is: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 30s, annealing at 57 - 63℃ for 30s, extension at 72℃ for 30s, with 35 cycles; Final extension at 72℃ for 8min.

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