CN113881782A - Primer group, kit, identification method and application for identifying sex of macrobrachium rosenbergii - Google Patents
Primer group, kit, identification method and application for identifying sex of macrobrachium rosenbergii Download PDFInfo
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Abstract
The application discloses a primer group for PCR amplification of sex chromosome fragments of macrobrachium rosenbergii, which comprises at least one pair of a first primer pair shown as SEQ ID NO. 1-2, a second primer pair shown as SEQ ID NO. 3-4 and a third primer pair shown as SEQ ID NO. 5-6. The primer group can effectively amplify specific fragments, can effectively identify the sex of the macrobrachium rosenbergii according to the fragments, and even can distinguish male, female and super-female genotypes, thereby providing help for cross breeding and genotyping of the macrobrachium rosenbergii.
Description
Technical Field
The application relates to the technical field of macrobrachium rosenbergii sex chromosome molecular identification, in particular to a primer group, a kit, an identification method and application for identifying the sex of macrobrachium rosenbergii.
Background
Macrobrachium rosenbergii (Macrobrachium rosenbergii) is a large freshwater shrimp belonging to the genus Macrobrachium of the family Macrobrachium of the order decapod of the Crustacea of the phylum Arthropoda. It is an important economic shrimp widely distributed in south and southeast Asia as well as in the Western Pacific region. The growth and development rates of the male and female individuals are obviously different, after sexual maturity, the male individuals are obviously larger than the female individuals, and the economic effect is higher. The method is an effective strategy for improving the yield by cultivating the macrobrachium rosenbergii of the full male population. And the yield of the parthenocarpy culture is higher than that of the conventional male and female mixed culture, so that the economic benefit of the culture can be greatly improved by utilizing the parthenocarpy culture.
In crustaceans, the androgenic gland is a key organ specific to males and plays a key role in the development of male gonads and secondary sexual characteristics of crustaceans (Sagi A, Dan C, Milner Y.Effect of sexual gland formation on physiological differentiation and sexual characteristics of crustaceans, macro fresh waters prawns, Macrobrachium rosenbergii [ J ] Gen.Comp.Endocrinol.1990,77(1): 15-22.). The sex differentiation direction can be controlled by cutting or transplanting the androgenic gland, which is reported in the research of the sex differentiation of the macrobrachium rosenbergii. The androgenic gland of male macrobrachium is removed to obtain feminized individuals. Such individuals can be mated with normal male individuals to obtain a fully male population of Macrobrachium rosenbergii (Aflalo ED, Honng TTT, Nguyen VH, et al. A novel two-step procedure for a mass production of all-mass publications of the giant fresh water prawn Macrobrachium rosenbergii [ J ]. Aquaculture.2006,256(1):468 and 478.).
However, the method has very important significance for accurately identifying the sex of the macrobrachium rosenbergii in the cross breeding process of the macrobrachium rosenbergii. However, macrobrachium rosenbergii, as a decapod animal, has a large number of chromosomes, a small sex chromosome, and is difficult to type. Therefore, it is critical to establish a method for efficiently amplifying the sex chromosome DNA.
Disclosure of Invention
In view of the above, the present application aims to provide at least a method for effectively amplifying sex chromosome DNA of macrobrachium rosenbergii, and the method can effectively identify the sex of the macrobrachium rosenbergii, so as to be applied to cross breeding and genotyping of the macrobrachium rosenbergii.
In a first aspect, the embodiment of the application discloses a primer group for PCR amplification of sex chromosome fragments of macrobrachium rosenbergii, which comprises at least one pair of a first primer pair shown as SEQ ID NO. 1-2, a second primer pair shown as SEQ ID NO. 3-4 and a third primer pair shown as SEQ ID NO. 5-6.
In the embodiment of the present application, the target sequence of the first primer pair is shown as SEQ ID No.7, the target sequence of the second primer pair is shown as SEQ ID No.8, and the target sequence of the third primer pair is shown as SEQ ID No. 9.
In a second aspect, the embodiment of the application discloses a kit for identifying the sex of macrobrachium rosenbergii, which comprises at least one pair of a first primer pair shown as SEQ ID No. 1-2, a second primer pair shown as SEQ ID No. 3-4 and a third primer pair shown as SEQ ID No. 5-6.
In a third aspect, the embodiment of the application discloses a method for identifying the sex of macrobrachium rosenbergii, which comprises the following steps:
extracting the genome DNA of the muscle tissue of the macrobrachium rosenbergii;
carrying out PCR amplification on the genomic DNA, and carrying out PCR amplification on at least one of a first primer pair shown as SEQ ID NO. 1-2, a second primer pair shown as SEQ ID NO. 3-4 and a third primer pair shown as SEQ ID NO. 5-6 to obtain an amplification product;
detecting the amplification product, and determining the sex of the macrobrachium rosenbergii according to the detection result.
In the present embodiment, the genomic DNA is amplified using the first primer pair, and the amplified product is electrophoretically detected; if the electrophoresis band comprises a 545bp band, the macrobrachium rosenbergii is male; if the electrophoretic band includes a 545bp band and a 390bp band, the macrobrachium rosenbergii is female.
In the present embodiment, the genomic DNA is amplified using the second primer pair, and the amplified product is electrophoretically detected; if the electrophoresis band comprises a 119bp band, the macrobrachium rosenbergii is female; if the electrophoresis band is not amplified, the macrobrachium rosenbergii is male.
In the present example, the genomic DNA was amplified using the second primer set, and the amplification product was quantitatively analyzed, and the macrobrachium rosenbergii corresponding to the sample with a large relative expression level was a super-female, the macrobrachium rosenbergii corresponding to the sample with a small relative expression level was a female, and the macrobrachium rosenbergii corresponding to the sample without an expression level was a male individual.
In the present example, the genomic DNA was amplified using the third primer set, and the amplification product was quantitatively analyzed, and the macrobrachium rosenbergii corresponding to the sample with a large relative expression level was male, the macrobrachium rosenbergii corresponding to the sample with a small relative expression level was female, and the macrobrachium rosenbergii corresponding to the sample without an expression level was a super-female individual.
In the examples of the present application, the amplification system for PCR amplification comprises: 8.5. mu.L of Mix system, 0.25. mu.L each of 10. mu.M upstream and downstream primers, and 1. mu.L of 100 ng/. mu.L genomic DNA as a template; wherein the Mix system comprises 100mM KCL, 20mM Tris-CL and 3mM MgCL20.4mM dNTP mix, 0.1U/. mu.LTaq DNA polymerase;
the PCR amplification program comprises the steps of carrying out 95 ℃ treatment for 5min, 95 ℃ treatment for 30s and 60 ℃ treatment for 30s in sequence, and continuing to carry out 72 ℃ treatment for 30s and 72 ℃ treatment for 10min in sequence after 30 cycles.
In a fourth aspect, the embodiments of the present application disclose the use of the primer set of the first aspect and the kit of the second aspect in sex determination, cross breeding or genotyping of macrobrachium rosenbergii.
Compared with the prior art, the application has at least the following beneficial effects:
the embodiment of the application provides a primer group and a method for amplifying sex chromosome segments of macrobrachium rosenbergii, the primer group can effectively amplify specific segments, the sex of the macrobrachium rosenbergii can be effectively identified according to the segments, even the male, female and super-female genotypes can be distinguished, and the primer group provides help for cross breeding and genotyping of the macrobrachium rosenbergii.
Drawings
Fig. 1 is a graph of HE staining of the gonadal tissue of macrobrachium rosenbergii provided in the examples of the present application.
FIG. 2 is an electrophoresis chart of the extracted DNA from the gonad tissue of Macrobrachium rosenbergii, the first lane is Marker, and the others are extracted DNA samples from different individual Macrobrachium rosenbergii.
FIG. 3 shows the electrophoresis result of the PCR amplification products of the male and female Macrobrachium rosenbergii with the first primer provided in the examples.
FIG. 4 shows the result of electrophoresis of PCR amplification products of the macrobrachium rosenbergii male and female individuals with the second primer provided in the examples of the present application.
FIG. 5 shows the quantitative results of (A) the qPCR for the DNA of the second primer pair to amplify the super female shrimp, the normal female shrimp and the male shrimp and (B) the qPCR for the DNA of the third primer pair to amplify the super female shrimp, the normal female shrimp and the male shrimp.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. The reagents or instruments referred to in the following examples are available from commercial gumming, unless otherwise specified; unless otherwise specified, the experimental methods are all conventional methods.
1. Macrobrachium rosenbergii and tissue material
8 female macrobrachium rosenbergii are purchased from orchard farmer markets, muscle tissues of the macrobrachium rosenbergii are immediately frozen by liquid nitrogen, and the macrobrachium rosenbergii is stored in a refrigerator at the temperature of-80 ℃ for later use.
Identifying the male and female phenotype of the macrobrachium rosenbergii: adopting macrobrachium rosenbergii gonad tissues, embedding, slicing and carrying out conventional HE dyeing, and observing the gonad slices under a microscope to determine male and female, wherein A is shown in figure 1; female ovarian section, B is male testis section.
2. Extraction of DNA:
1) approximately 0.2g of frozen muscle tissue was placed in a 1.5mL EP tube, the muscle was minced with sterile scissors, 600. mu.L of lysis buffer was added, and the tube was digested in a 55 ℃ water bath until the muscle was completely dissolved, with the period being reversed several times.
2) After the digest was cooled to room temperature, 2. mu.L of LRNAnase (10mg/ml) was added to each tube and digested at 37 ℃ for 30 min.
3) Adding an equal volume of saturated phenol solution, shaking up and down for 15min, and centrifuging at 12000rpm for 15min at 4 ℃.
4) The supernatant was pipetted into a new EP tube and an equal volume of phenol was added: chloroform: isoamyl alcohol (25:24:1), shaking upside down for 15min, and centrifuging at 12000rpm for 15min at 4 ℃.
5) The supernatant was pipetted into a fresh EP tube and an equal volume of chloroform was added: isoamyl alcohol (24:1), shaking upside down for 15min, and centrifuging at 12000rpm for 5min at 4 ℃.
6) The supernatant was pipetted into a fresh EP tube and two volumes of pre-cooled absolute ethanol were added to the supernatant. The tube was rotated upside down to precipitate the DNA. Centrifuging at 4 deg.C for 10min at 12,000 rpm
7) The supernatant was discarded, and the precipitate was washed with 1ml of 70% absolute ethanol and centrifuged at 8000rpm for 10min at 4 ℃.
8) Sucking off ethanol, opening the cover, standing at room temperature for drying for 10min, adding 50 μ L of non-enzyme water to dissolve precipitate
And (3) carrying out electrophoresis on the extracted DNA in 1.5% agarose gel, and detecting that the A260/280 value of the DNA is between 1.8 and 2.0 by using an enzyme-linked immunosorbent assay. Subsequent experiments were performed with good integrity of the DNA, such as the DNA shown in fig. 2.
2. First primer pair
The application further sequences the extracted DNA fragments, selects fragments related to sex as target sequences, and designs a first primer pair according to the target sequences.
The nucleotide sequence of the target sequence is shown as SEQ ID NO.7, and the nucleotide sequence of the first primer pair is shown as SEQ ID NO. 1-2, which are provided by Shanghai chemical company.
3. PCR amplification Using the first primer pair
PCR amplification was carried out using the DNA fragments (about 100 ng/. mu.L) obtained from 16 macrobrachium rosenbergii extracted as described above as amplification templates.
The amplification system consisted of 20. mu.L: mix 10. mu.L, F primer 0.25. mu.L, R primer 0.25. mu.L, template 1. mu.L and ddH2O 8.5μL。
The amplification procedure was: 95 ℃ for 5min → 95 ℃ for 30s → 60 ℃ for 30 s; 30 cycles; → 72 deg.C for 30s → 72 deg.C for 10 min.
The amplification results are shown in FIG. 3. The DNA amplification results of 8 female individuals have two bands, and the DNA amplification results of 8 male individuals only have one band, so that the sex of the macrobrachium rosenbergii can be identified according to the number of the bands by carrying out PCR amplification on the muscle tissues of the macrobrachium rosenbergii and carrying out electrophoresis detection on the amplification products according to the first primer pair.
4. Amplified band sequencing
In the results of the above examples, the band with a larger molecular weight was a male-female common band, and the band with a smaller molecular weight was a female-specific band. The DNA sequence of the female-specific band was named as the first sequence, the DNA sequence of the male-female common band was named as the second sequence, and sequencing and multiple alignment of 3 sequences revealed that the homology of the first sequence (shown as SEQ ID NO. 8) and the second sequence (shown as SEQ ID NO. 9) was 83.42%.
5. Specific primers for first and second sequences
According to the difference of the first sequence and the second sequence found by the comparison result, a second primer pair aiming at the first sequence and a third primer pair aiming at the second sequence are respectively designed, the nucleotide sequence of the second primer pair is shown as SEQ ID NO. 3-4, and the nucleotide sequence of the third primer pair is shown as SEQ ID NO. 5-6. The second primer pair and the third primer pair are both provided by Shanghai Biotech.
Experimental materials: healthy 16 sexually mature female macrobrachium rosenbergii and 16 male macrobrachium rosenbergii from the vegetable market, local fisheries markets in china, vietnam and bangladesh, and 83 super females from one farming farm in Yangzhou (imported from israel). Wherein the androgenic gland is transplanted into a female individual to cause the female to become sexually inverted into a male, and the individual is mated with a normal female to produce offspring having a ratio of male to female of 3:1, wherein the offspring comprises a super female.
Extracting the genome DNA of the muscle tissue of the macrobrachium rosenbergii by the same method, amplifying by the same PCR amplification method, and performing population verification on 16 female individuals and 16 male individuals by using a second primer pair. As shown in FIG. 4, a band 119bp long was amplified in 16 female individuals, while no band was amplified in any male individual. This demonstrates that the second primer pair provided in the examples of the present application has specificity for the first sequence and is not capable of amplifying the second sequence. And the second primer pair is used for PCR amplification and electrophoresis detection, and the sex of the macrobrachium rosenbergii can be judged according to an electrophoresis strip.
And similarly, the second primer pair or the third primer pair is used for carrying out qPCR amplification on the super-female individual, the female individual and the male individual, and the sex of the macrobrachium rosenbergii can also be judged according to the quantitative result of the qPCR on the amplification product. The specific process is as follows:
DNA of the above experimental material having the group DNA was extracted by the same method as described above. Primers that specifically amplified the chromosome fragments were then tested by qPCR to determine dose differences between the super female individuals, female individuals and male individuals.
The qPCR reaction system (20. mu.L) contained 10. mu.L of SYRBgreen (TaKaRa), 1. mu.L of DNA template, 0.25. mu.L of each of the upstream and downstream primers, and 8.5. mu.L of water. The PCR cycle parameters were as follows: 95 ℃ 3min → 95 ℃ 30s (40 cycles) → 60 ℃ 30s → 72 ℃ 30s → 72 ℃ extension for 10min, with the primer of β -actin as the internal control. By using 2－△△CTRelative expression levels were calculated and run in triplicate for each sample.
The results are shown in FIG. 5. As shown in FIG. 5A, the amplification results of the second primer set revealed that the superfemale individuals with a large relative expression level were normal female individuals with a small relative expression level, and that the superfemale individuals were twice as large relative expression levels as the normal female individuals; while no expression was found to be male.
Similarly, as shown in FIG. 5B, the amplification results of the third primer pair revealed that the male individuals with relatively large expression level and the normal female individuals with relatively small expression level were male individuals with relatively large expression level, and the relative expression levels of the male individuals and the normal female individuals had a double dose relationship; while those with no expression were hyperfemale individuals. Therefore, the sex identification of the macrobrachium rosenbergii can be realized by the amplification of the third primer pair.
Therefore, the second primer pair disclosed in the examples of the application can be used for distinguishing the super female shrimp from the normal female shrimp, and the third primer pair can be used for distinguishing the female shrimp from the male shrimp and also can be used for relative expression quantity, so that a brand-new method is provided for sex identification of the macrobrachium rosenbergii.
In summary, the specific segment related to the sex chromosome of the macrobrachium rosenbergii can be amplified through the primer group, so that the sex of the macrobrachium rosenbergii can be identified, and the method provides help for obtaining the whole male individual breeding of the macrobrachium rosenbergii; and female and super-female macrobrachium rosenbergii individuals can be accurately identified through the specific primers, so that the method provides help for cross breeding and genotyping research of the macrobrachium rosenbergii individuals.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Sequence listing
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cctac 545
Claims (10)
1. The primer group for PCR amplification of the sex chromosome segment of the macrobrachium rosenbergii comprises at least one pair of a first primer pair shown as SEQ ID NO. 1-2, a second primer pair shown as SEQ ID NO. 3-4 and a third primer pair shown as SEQ ID NO. 5-6.
2. The primer set of claim 1, wherein the target sequence of the first primer pair is shown as SEQ ID No.7, the target sequence of the second primer pair is shown as SEQ ID No.8, and the target sequence of the third primer pair is shown as SEQ ID No. 9.
3. A kit for identifying the sex of macrobrachium rosenbergii comprises at least one pair of a first primer pair shown as SEQ ID No. 1-2, a second primer pair shown as SEQ ID No. 3-4 and a third primer pair shown as SEQ ID No. 5-6.
4. A method for identifying the sex of macrobrachium rosenbergii comprises the following steps:
extracting the genome DNA of the muscle tissue of the macrobrachium rosenbergii;
carrying out PCR amplification on the genomic DNA, and carrying out PCR amplification on at least one of a first primer pair shown as SEQ ID NO. 1-2, a second primer pair shown as SEQ ID NO. 3-4 and a third primer pair shown as SEQ ID NO. 5-6 to obtain an amplification product;
detecting the amplification product, and determining the sex of the macrobrachium rosenbergii according to the detection result.
5. The method for sex determination according to claim 4, wherein the genomic DNA is amplified using the first primer pair, and the amplified product is electrophoretically detected;
if the electrophoresis band comprises a 545bp band, the macrobrachium rosenbergii is male; if the electrophoretic band includes a 545bp band and a 390bp band, the macrobrachium rosenbergii is female.
6. The method for sex determination according to claim 4, wherein the genomic DNA is amplified using the second primer pair, and the amplified product is electrophoretically detected;
if the electrophoresis band comprises a 119bp band, the macrobrachium rosenbergii is female; if the electrophoresis band is not amplified, the macrobrachium rosenbergii is male.
7. The method of identifying sex according to claim 4, wherein the genomic DNA is amplified using the second primer set, and the amplification product is quantitatively analyzed, and wherein macrobrachium rosenbergii corresponding to a sample with a large relative expression level is a super-female, macrobrachium rosenbergii corresponding to a sample with a small relative expression level is a female, and macrobrachium rosenbergii corresponding to a sample with no expression level is a male individual.
8. The method of identifying sex according to claim 4, wherein the genomic DNA is amplified using the third primer set, and the amplification product is quantitatively analyzed, wherein macrobrachium rosenbergii corresponding to a sample with a large relative expression level is male, macrobrachium rosenbergii corresponding to a sample with a small relative expression level is female, and macrobrachium rosenbergii corresponding to a sample with no expression level is a super-female individual.
9. The method for sex determination according to claim 4, wherein the PCR-amplified amplification system comprises: 8.5. mu.L of Mix system, 0.25. mu.L each of 10. mu.M upstream and downstream primers, and 1. mu.L of 100 ng/. mu.L genomic DNA as a template; wherein the Mix system comprises 100mM KCL, 20mM Tris-CL and 3mM MgCL20.4mM dNTP mix, 0.1U/. mu.LTaq DNA polymerase;
the PCR amplification program comprises the steps of carrying out 95 ℃ treatment for 5min, 95 ℃ treatment for 30s and 60 ℃ treatment for 30s in sequence, and continuing to carry out 72 ℃ treatment for 30s and 72 ℃ treatment for 10min in sequence after 30 cycles.
10. The primer set according to claim 1 or 2 and the kit according to claim 3 are used for sex determination, cross breeding or genotyping of macrobrachium rosenbergii.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015034347A1 (en) * | 2013-09-09 | 2015-03-12 | Universiti Malaya | A method of identifying parentage in freshwater prawn macrobrachium rosenbergii |
| CN111996261A (en) * | 2020-07-31 | 2020-11-27 | 湖南银鱼农业科技有限公司 | Macrobrachium rosenbergii sex molecular marker primer and application thereof |
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- 2021-10-25 CN CN202111241232.2A patent/CN113881782A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015034347A1 (en) * | 2013-09-09 | 2015-03-12 | Universiti Malaya | A method of identifying parentage in freshwater prawn macrobrachium rosenbergii |
| CN111996261A (en) * | 2020-07-31 | 2020-11-27 | 湖南银鱼农业科技有限公司 | Macrobrachium rosenbergii sex molecular marker primer and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XUE-HUI JIANG等: "Female-only sex-linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii", ANIMAL GENETICS, vol. 44, no. 6, pages 782 - 785, XP055585073, DOI: 10.1111/age.12067 * |
| 姜建萍;袁翔;邱庆庆;黄光华;杨秀荣;蒋和生;杨学明;蒋钦杨;: "罗氏沼虾TRY基因生物信息学及其mRNA表达分析", 西南农业学报, no. 06, pages 1327 - 1331 * |
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