PCR (Polymerase Chain Reaction) amplification primer, method and kit for quickly distinguishing genetic sex of Chinese softshell turtle

Abstract

The invention discloses a PCR (Polymerase Chain Reaction) amplification primer for quickly distinguishing the genetic sex of a Chinese softshell turtle. The PCR amplification primer comprises a pair of sex specificity molecular markers and a pair of reference gene primers. The invention further discloses a PCR method for quickly distinguishing the genetic sex of the Chinese softshell turtle and akit for quickly distinguishing the genetic sex of the Chinese softshell turtle. The amplification primer can distinguish the genetic sex of the Chinese softshell turtle in the early stage. The methodis low in cost and easy and convenient to operate and can quickly distinguish a large batch of turtles, and a result is intuitive and accurate.

Description

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a PCR amplification primer, a method and a kit for quickly identifying the genetic sex of Chinese soft-shelled turtle. Background technique

[0002] Chinese soft-shelled turtle (Pelodiscus sinensis), belonging to the order Chelonia, is the oldest group of tetrapods, and it is the junction point for the evolution of amniotic amphibians to amniotic egg-laying reptiles. It has unique scientific research value in the fields of studying species morphological evolution and reproductive mode evolution. With the rapid development of the turtle breeding industry, it has added its pivotal economic value. In 2016, the total national output of Chinese soft-shelled turtle reached more than 340,000 tons. It is a famous, special and high-quality aquaculture species with high economic value, and it is an important part of freshwater aquaculture.

[0003] For a long time...

Examples

Embodiment 1

[0037] The PCR amplification primers for quickly identifying the genetic sex of soft-shelled turtle provided in this example include a pair of sex-specific molecular marker primers and a pair of internal reference gene primers, wherein:

[0038] The sequence of the upstream primer of the sex-specific molecular marker primer is shown in SEQ ID NO: 1;

[0039] The sequence of the downstream primer of the sex-specific molecular marker primer is shown in SEQ ID NO: 2;

[0040] The sequence of the upstream primer of the internal reference gene primer is shown in SEQ ID NO: 3;

[0041] The sequence of the downstream primer of the internal reference gene primer is shown in SEQ ID NO:3.

[0042] specific:

[0043] The sequence of the upstream primer of the sex-specific molecular marker primer is:

[0044] GTGGAAACAATATCATCGCCGAG.

[0045] The sequence of the downstream primer of the sex-specific molecular marker primer is:

[0046] TGTGTGCCGTGCCTGCGAAGC.

[0047] The sequence of...

Embodiment 2

[0051] The PCR method for quickly identifying the genetic sex of Chinese soft-shelled turtle provided by the present embodiment comprises the following steps

[0052] (1) Tissue collection of the individual under examination

[0053] A total of 200 Chinese soft-shelled turtles were randomly selected. After the sex was detected by gonad anatomy and histology, the tissues of each individual were cut, soaked in absolute ethanol and stored at -20°C until use;

[0054] (2) Extraction of genomic DNA

[0055] Before DNA extraction, toenails were soaked in 1×PBS buffer at 4°C overnight, and washed 2-3 times during this period. Take about 5-20 mg of each toenail sample, use the MicroElute Genomic DNA Kit (OMEGA, USA) kit to extract DNA according to the instructions, and store the extracted DNA at -20°C until use;

[0056] Specific steps are as follows:

[0057] ①. Add the toenail sample to a 1.5mL centrifuge tube, and add 210-220μL of TL Buffer;

[0058] ②. Cut the toenail sample a...

Embodiment 3

[0083] In this example, DNA was extracted from 200 randomly selected soft-shelled turtles, PCR amplification was performed using internal reference gene primers and sex-specific molecular marker primers, and after agarose gel electrophoresis and ultraviolet light detection, the obtained individual The genetic sex data (female:male is 105:95) is consistent with the sex data of gonad anatomy and histology (female:male is 105:95), and the identification accuracy rate is 100%.

[0084] Therefore, the PCR amplification primers for quickly identifying the genetic sex of Chinese soft-shelled turtle in the present invention can be further developed into a kit for quickly identifying the genetic sex of Chinese soft-shelled turtle.

[0085] The present invention adopts PCR technology for the first time as a method for quick identification of the genetic sex of Chinese soft-shelled turtle (especially juvenile soft-shelled turtle), and uses a pair of Chinese soft-shelled turtle genetic sex...

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a PCR amplification primer, a method and a kit for quickly identifying the genetic sex of Chinese soft-shelled turtle. Background technique

[0002] Chinese soft-shelled turtle (Pelodiscus sinensis), belonging to the order Chelonia, is the oldest group of tetrapods, and it is the junction point for the evolution of amniotic amphibians to amniotic egg-laying reptiles. It has unique scientific research value in the fields of studying species morphological evolution and reproductive mode evolution. With the rapid development of the turtle breeding industry, it has added its pivotal economic value. In 2016, the total national output of Chinese soft-shelled turtle reached more than 340,000 tons. It is a famous, special and high-quality aquaculture species with high economic value, and it is an important part of freshwater aquaculture.

[0003] For a long time...

Examples

Embodiment 1

[0037] The PCR amplification primers for quickly identifying the genetic sex of soft-shelled turtle provided in this example include a pair of sex-specific molecular marker primers and a pair of internal reference gene primers, wherein:

[0038] The sequence of the upstream primer of the sex-specific molecular marker primer is shown in SEQ ID NO: 1;

[0039] The sequence of the downstream primer of the sex-specific molecular marker primer is shown in SEQ ID NO: 2;

[0040] The sequence of the upstream primer of the internal reference gene primer is shown in SEQ ID NO: 3;

[0041] The sequence of the downstream primer of the internal reference gene primer is shown in SEQ ID NO:3.

[0042] specific:

[0043] The sequence of the upstream primer of the sex-specific molecular marker primer is:

[0044] GTGGAAACAATATCATCGCCGAG.

[0045] The sequence of the downstream primer of the sex-specific molecular marker primer is:

[0046] TGTGTGCCGTGCCTGCGAAGC.

[0047] The sequence of...

Embodiment 2

[0051] The PCR method for quickly identifying the genetic sex of Chinese soft-shelled turtle provided by the present embodiment comprises the following steps

[0052] (1) Tissue collection of the individual under examination

[0053] A total of 200 Chinese soft-shelled turtles were randomly selected. After the sex was detected by gonad anatomy and histology, the tissues of each individual were cut, soaked in absolute ethanol and stored at -20°C until use;

[0054] (2) Extraction of genomic DNA

[0055] Before DNA extraction, toenails were soaked in 1×PBS buffer at 4°C overnight, and washed 2-3 times during this period. Take about 5-20 mg of each toenail sample, use the MicroElute Genomic DNA Kit (OMEGA, USA) kit to extract DNA according to the instructions, and store the extracted DNA at -20°C until use;

[0056] Specific steps are as follows:

[0057] ①. Add the toenail sample to a 1.5mL centrifuge tube, and add 210-220μL of TL Buffer;

[0058] ②. Cut the toenail sample a...

Embodiment 3

[0083] In this example, DNA was extracted from 200 randomly selected soft-shelled turtles, PCR amplification was performed using internal reference gene primers and sex-specific molecular marker primers, and after agarose gel electrophoresis and ultraviolet light detection, the obtained individual The genetic sex data (female:male is 105:95) is consistent with the sex data of gonad anatomy and histology (female:male is 105:95), and the identification accuracy rate is 100%.

[0084] Therefore, the PCR amplification primers for quickly identifying the genetic sex of Chinese soft-shelled turtle in the present invention can be further developed into a kit for quickly identifying the genetic sex of Chinese soft-shelled turtle.

[0085] The present invention adopts PCR technology for the first time as a method for quick identification of the genetic sex of Chinese soft-shelled turtle (especially juvenile soft-shelled turtle), and uses a pair of Chinese soft-shelled turtle genetic sex...

Images