A breeding method for maintaining excellent growth traits of naked carp

Abstract

The present invention belongs to the field of animal molecular breeding, and relates to a breeding method capable of maintaining excellent growth traits of sharp naked carp, the breeding method comprising 1) selecting parents; 2) identifying the kinship of the parents and pairing the parents; 3) culturing the paired parents to obtain the first generation of sharp naked carp; 4) selecting reserve parents from all the first generation of sharp naked carp; 5) identifying the kinship of the first generation of sharp naked carp and pairing the parents of the first generation of sharp naked carp; 6) culturing the paired first generation of sharp naked carp parents to obtain the second generation of sharp naked carp, which is a sharp naked carp capable of maintaining excellent growth traits of sharp naked carp. The present invention provides a breeding method capable of maintaining excellent growth traits of sharp naked carp, which can avoid inbreeding of sharp naked carp during artificial reproduction, can carry out large-scale production, and provide technical support for artificial release and germplasm preservation of sharp naked carp.

Description

Technical Field

[0001] The invention belongs to the field of animal molecular breeding, relates to a breeding method for sharp naked carp, and in particular to a breeding method capable of maintaining excellent growth traits of sharp naked carp. Background Art

[0002] Oxygymnocypris stewartii belongs to the Cypriniformes, Cyprinidae, Schizothorinae, Oxygymnocypris stewartii, commonly known as Stewart's naked carp. It lives in plateau waters above 3,600 meters above sea level, and mostly moves in the flowing water of major tributaries in the middle reaches of the river. Due to the high altitude and low annual average water temperature, a special ecological environment has been formed. Oxygymnocypris adapts to an environment with clear water sources such as precipitation and mountain snow water. It mainly feeds on other fish and aquatic insects. Oxygymnocypris stewartii is endemic to my country and is only distributed in the middle reaches of the Yarlung Zangbo River. It is often found in the flowing water of the main and tributary rivers. The origin of this species is Nianchu River.

[0003] At present, there are few research materials on the sharp naked carp. In the early days, most of them were studies on taxonomy, origin and evolution, chromosome number and karyotype. In recent years, they have focused on molecular phylogeny and biogeography. There are few reports on the reproduction and breeding of sharp naked carp. At present, crayfish, four major carps and other fish are facing the problem of inbreeding, and sharp naked carp is no exception. The population of sharp naked carp is small, and wild individuals are difficult to catch. It is difficult for sharp naked carp breeding bases to avoid inbreeding between individuals when carrying out artificial breeding of sharp naked carp. Inbreeding will lead to the degeneration of sharp naked carp populations and the problem that sharp naked carp individuals are not resistant to diseases and grow slowly. Therefore, it is urgent to select and breed sharp naked carp. Summary of the invention

[0004] In order to solve the above-mentioned technical problems existing in the background technology, the present invention provides a breeding method that can avoid inbreeding of sharp naked carp during artificial breeding, can carry out large-scale production, and provide technical support for the artificial release and germplasm preservation of sharp naked carp, which can maintain the excellent growth traits of sharp naked carp.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A microsatellite marker primer for naked carp, characterized in that: the microsatellite marker primer for naked carp comprises 5 groups of microsatellite marker primers for naked carp combined with a double PCR system, specifically comprising 10 pairs of microsatellite primers, and the sequences and annealing temperatures of the 10 pairs of microsatellite primers are respectively:

[0007]

[0008]

[0009] Based on the above-mentioned application of microsatellite marker primers for naked carp in the breeding of naked carp.

[0010] Preferably, the microsatellite marker primers for sharp naked carp provided by the present invention are used in the breeding of sharp naked carp with excellent growth traits.

[0011] A breeding method capable of maintaining excellent growth traits of sharp naked carp, characterized in that the breeding method capable of maintaining excellent growth traits of sharp naked carp comprises the following steps:

[0012] 1) Selecting parents; the parents are naked carp with excellent traits collected from farms and the wild; the excellent traits are fast growth and strong immunity (strong immunity refers to traits, virus tolerance);

[0013] 2) performing kinship identification and parent pairing on the parents selected in step 1);

[0014] 3) culturing the paired parents obtained in step 2) to obtain the first generation naked carp;

[0015] 4) Selecting backup parents from all the next generation naked carp;

[0016] 5) performing genetic relationship identification of the next-generation naked carp and parent pairing of the next-generation naked carp on the backup parents selected in step 4);

[0017] 6) Cultivating the paired first-generation naked carp parents obtained according to step 5) to obtain second-generation naked carp, wherein the second-generation naked carp can maintain the excellent growth traits of naked carp.

[0018] Preferably, the specific implementation of step 2) adopted in the present invention is:

[0019] 2.1) PIT marking is performed on the parents selected in step 1);

[0020] 2.2) using the microsatellite marker primers of naked carp to establish a phylogenetic clustering tree for the parents of step 2.1);

[0021] 2.3) According to the kinship clustering tree constructed in step 2.2), individuals that are not in the same branch of the clustering tree are selected for pairing and breeding to construct different pairing and breeding pedigrees.

[0022] Preferably, the specific implementation method of step 3) adopted by the present invention is:

[0023] 3.1) artificially incubating the offspring produced by breeding the paired parents obtained in step 2);

[0024] 3.2) After hatching, the first generation naked carp were cultured separately under the same conditions according to different families. The first generation naked carp were fed with commercial feed three times a day, and the feeding amount was 0.5% of the body weight of the first generation naked carp.

[0025] Preferably, the specific implementation method of step 4) adopted by the present invention is:

[0026] 4.1) After the first generation of naked carp obtained in step 3) is cultured for one year, the body length and weight of all naked carp in each family are measured, and the first generation of naked carp with excellent traits are selected; the excellent traits are fast growth rate and strong immunity;

[0027] 4.2) The selected first generation naked carp with excellent traits are cultured to sexual maturity;

[0028] 4.3) Select the first-generation female and the first-generation male naked carp from each family as backup parents.

[0029] Preferably, the specific implementation of step 5) adopted by the present invention is:

[0030] 5.1) PIT marking is performed on the backup parents selected in step 4);

[0031] 5.2) Using the microsatellite marker primers of naked carp to establish a phylogenetic clustering tree for the parents of step 5.1);

[0032] 5.3) According to the phylogenetic cluster development tree constructed in step 2.2), the first generation naked carp that are not in the same branch of the cluster development tree are selected for pairing and breeding.

[0033] Preferably, the specific implementation method of the kinship identification in step 2) and step 5) adopted in the present invention is: taking a tissue sample of the naked carp to be identified; extracting DNA of the naked carp sample; using 5 groups of naked carp microsatellite marker primers combined with a double PCR system to perform PCR amplification on the naked carp sample; performing electrophoresis separation on a 10% polyacrylamide gel on the PCR amplification products; statistically calculating the genotypes of the PCR amplification products based on the separation results; and using Mega software to establish a kinship cluster development tree based on the genotyping results of each individual.

[0034] Preferably, the reaction system of PCR amplification in the kinship identification used in the present invention is 25ul: 10×PCRBuffer 2.5ul, 2.5mmol / L dNTP 1.5ul, 2mmol / L MgCl2 2.5ul, upstream and downstream primers 1ul each, 0.5U / μLTaq enzyme 1ul, DNA template 2ul, ultrapure water 11.5ul;

[0035] The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at annealing temperature for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The invention can avoid the phenomenon of inbreeding caused by blind pairing in traditional artificial breeding of sharp naked carp. The invention can mass-produce sharp naked carp with fast growth rate and strong immunity. The invention provides technical support for artificial release and germplasm preservation of sharp naked carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a result diagram of the phylogenetic relationships of 100 naked carp to be identified in the embodiment;

[0039] Figure 2 It is a diagram showing the phylogenetic relationships of 48 naked carp to be identified in the embodiment. DETAILED DESCRIPTION

[0040] The present invention provides a microsatellite marker primer for naked carp, including 5 groups of microsatellite marker primer combination double PCR systems for naked carp, specifically including 10 pairs of microsatellite primers, and the sequences and annealing temperatures of the 10 pairs of microsatellite primers are respectively:

[0041]

[0042] Based on the application of the aforementioned microsatellite marker primers for sharp naked carp in the breeding of sharp naked carp, especially in the breeding of sharp naked carp with excellent growth traits.

[0043] The present invention also provides a breeding method capable of maintaining excellent growth traits of naked carp, the method comprising the following steps:

[0044] 1) Select parents; the parents are sharp naked carp with excellent traits collected from farms and the wild; the excellent traits are fast growth and strong immunity; for example, 50 sharp naked carp with fast growth and strong immunity can be collected from different sharp naked carp farms. 50 sharp naked carp with fast growth and strong immunity can be collected from the wild.

[0045] 2) Conducting kinship identification and parent pairing on the parents selected in step 1), specifically:

[0046] 2.1) PIT marking is performed on the parents selected in step 1);

[0047] 2.2) using the microsatellite marker primers of naked carp to establish a phylogenetic clustering tree for the parents of step 2.1);

[0048] 2.3) According to the kinship cluster development tree constructed in step 2.2), individuals that are not in the same branch of the cluster development tree are selected for pairing and breeding to construct different pairing and breeding pedigrees. For example, based on the 100 naked carps obtained above, 8-10 pedigrees can be constructed.

[0049] 3) Cultivating the paired parents obtained in step 2) to obtain the first generation naked carp, specifically:

[0050] 3.1) artificially incubating the offspring produced by breeding the paired parents obtained in step 2);

[0051] 3.2) After hatching, the first generation naked carp were cultured separately under the same conditions according to different families. The first generation naked carp were fed with commercial feed three times a day, and the feeding amount was 0.5% of the body weight of the first generation naked carp.

[0052] 4) Selecting backup parents from all the first generation naked carp, specifically:

[0053] 4.1) After the first generation of naked carp obtained in step 3) is cultured for one year, the body length and weight of all naked carp in each family are measured, and the first generation of naked carp with excellent traits are selected; the excellent traits are fast growth rate and strong immunity;

[0054] 4.2) The selected first generation naked carp with excellent traits are cultured to sexual maturity;

[0055] 4.3) Select the first generation female and the first generation male of G. acutifolia from each family as backup parents. For example, three female and male G. acutifolia from each family are selected as backup parents.

[0056] 5) The reserve parents selected in step 4) are subjected to genetic relationship identification of the first generation naked carp and parent pairing of the first generation naked carp, specifically:

[0057] 5.1) PIT marking is performed on the backup parents selected in step 4);

[0058] 5.2) Using the microsatellite marker primers of naked carp to establish a phylogenetic clustering tree for the parents of step 5.1);

[0059] 5.3) According to the phylogenetic cluster development tree constructed in step 2.2), the first generation naked carp that are not in the same branch of the cluster development tree are selected for pairing and breeding.

[0060] 6) Cultivating the paired first-generation naked carp parents obtained in step 5) to obtain second-generation naked carp, which can maintain the excellent growth traits of naked carp.

[0061] Among them, the specific implementation method of kinship identification in step 2) and step 5) is: taking a tissue sample of naked carp to be identified; extracting DNA of the naked carp sample; using 5 groups of naked carp microsatellite marker primers combined with a double PCR system to perform PCR amplification on the naked carp sample; performing electrophoresis separation on 10% polyacrylamide gel for PCR amplification products; counting the genotypes of the PCR amplification products according to the separation results; using Mega software to establish a kinship cluster development tree according to the genotyping results of each individual, and counting the kinship between each selected female fish and male fish.

[0062] The reaction system of PCR amplification in kinship identification adopted by the present invention is 25ul: 10×PCR Buffer 2.5ul, 2.5mmol / L dNTP 1.5ul, 2mmol / L MgCl2 2.5ul, upstream and downstream primers 1ul each, 0.5U / μL Taq enzyme 1ul, DNA template 2ul, ultrapure water 11.5ul; the reaction procedure of PCR amplification is: 94°C pre-denaturation for 3min; 94°C denaturation for 30s, annealing temperature renaturation for 30s, 72°C extension for 30s, 30 cycles; 72°C extension for 10min; 4°C storage.

[0063] The technical solution provided by the present invention will be described in detail below in conjunction with specific embodiments:

[0064] Example 1

[0065] 500 sharp naked carp were collected from different sharp naked carp breeding farms in the Yarlung Zangbo River Basin, and 500 wild sharp naked carp were collected from the wild. 50 artificially bred sharp naked carp with fast growth rate and strong immunity and 50 wild sharp naked carp were selected from the above sharp naked carp. The selected sharp naked carp were PIT marked and cultured in breeding ponds with suitable environments, marked as 1-50 and 51-100 respectively.

[0066] Take the above 100 samples of naked carp tissue to be identified; extract the DNA of the naked carp samples; use 5 groups of naked carp microsatellite marker primers to combine the double PCR system to perform PCR amplification on the naked carp samples; separate the PCR amplification products by electrophoresis on 10% polyacrylamide gel; count the genotypes of the PCR amplification products according to the separation results; use Mega software to establish a kinship clustering tree based on the genotyping results of each individual, and count the kinship between each selected female and male fish. The PCR reaction system is 25ul: 10×PCRBuffer2.5ul, 2.5mmol / LdNTP1.5ul, 2mmol / L MgCl22.5ul, 1ul of upstream and downstream primers, 1ul of 0.5U / μL Taq enzyme, 2ul of DNA template, and 11.5ul of ultrapure water. The PCR reaction program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at annealing temperature for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0067] According to the clustering tree Figure 1 It can be concluded that these 100 naked carp individuals are divided into 7 branches, that is, 7 large families. Individuals 1-10 and 91-100 are closely related and are divided into one family, individuals 51-70 are closely related and are divided into one family, individuals 21-30 and 71-80 are closely related and are divided into one family, individuals 31-40 are closely related and are divided into one family, individuals 81-90 are closely related and are divided into one family, individuals 41-50 are closely related and are divided into one family, and individuals 11-20 are closely related and are divided into one family. The above-mentioned individuals with close relationships cannot be paired for reproduction, but individuals from different branches can be paired for reproduction.

[0068] Male and female individuals were selected from the above 7 branches, and the male and female individuals from different branches were paired for reproduction. Individuals 9 and 80 were paired; individuals 31 and 11 were paired; individuals 59 and 41 were paired; individuals 85 and 12 were paired; individuals 49 and 29 were paired; individuals 70 and 79 were paired; individuals 30 and 69 were paired; and individuals 8 and 78 were paired.

[0069] The offspring of the sharp naked carp produced by the paired breeding of sharp naked carp in the previous step were artificially hatched. Each family was placed in a separate breeding pond and raised under the same conditions. The sharp naked carp were fed with commercial feed three times a day, and the feeding amount was 0.5% of the fish body weight. All the offspring of sharp naked carp were kept in the same living environment as much as possible. After one year of breeding, the body length and weight of all the sharp naked carp in each family were measured, and the sharp naked carp with fast growth rate and strong immunity were selected. When they were bred to sexual maturity, three sharp naked carp females and males were selected from each family as backup parents.

[0070] All the selected offspring of naked carp were PIT marked as 101-106, 107-112, 113-118, 119-124, 125-130, 131-136, 137-142, and 143-148 respectively.

[0071] Take the above 48 samples of naked carp tissue to be identified; extract the DNA of the naked carp samples; use 5 groups of naked carp microsatellite marker primers to combine the double PCR system to perform PCR amplification on the naked carp samples; separate the PCR amplification products by electrophoresis on 10% polyacrylamide gel; count the genotypes of the PCR amplification products according to the separation results; use Mega software to establish a kinship clustering tree based on the genotyping results of each individual, and count the kinship between each selected female and male fish. The PCR reaction system is 25ul: 10×PCRBuffer2.5ul, 2.5mmol / LdNTP1.5ul, 2mmol / L MgCl22.5ul, 1ul of upstream and downstream primers, 1ul of 0.5U / μLTaq enzyme, 2ul of DNA template, and 11.5ul of ultrapure water. The PCR reaction program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at annealing temperature for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0072] According to the clustering tree Figure 2 It can be concluded that these 48 sharp naked carp individuals are divided into 4 branches, that is, into 4 large families. Individuals 107-112 and 125-130 are closely related and are divided into one family. Individuals 113-118 and 137-142 are closely related and are divided into one family. Individuals 131-136 and 143-148 are closely related and are divided into one family. Individuals 101-106 and 119-124 are closely related and are divided into one family. Individuals with close relationships cannot be paired for reproduction, but individuals from different branches can be paired for reproduction. According to this cluster development book, the second generation of sharp naked carp produced by the pairing of the first generation of sharp naked carp was artificially hatched and cultured as mature individuals. The growth rate of this second generation of sharp naked carp is 10-20% faster than that of traditionally cultured sharp naked carp, and the second generation of sharp naked carp has a stronger immunity than traditionally cultured sharp naked carp. By using this method, a large number of naked carp with fast growth rate and strong immunity were obtained.

Claims

1. A microsatellite marker primer for naked carp, characterized in that: The microsatellite marker primers for the sharp naked carp include 5 groups of primers for double PCR system amplification, each group of primers includes 2 pairs of microsatellite primers, and the sequences and annealing temperatures of the microsatellite marker primers for the sharp naked carp are: 。 2. Application of the microsatellite marker primers for naked carp according to claim 1 in breeding of naked carp.

3. Application of the microsatellite marker primer of naked carp according to claim 2 in breeding of excellent growth traits of naked carp.

4. A breeding method capable of maintaining excellent growth traits of naked carp, characterized in that: The breeding method capable of maintaining excellent growth traits of naked carp comprises the following steps: 1) Selecting parents; the parents are naked carp with excellent traits collected from farms and the wild; the excellent traits are fast growth and strong immunity; 2) Conduct kinship identification and parent pairing for the parents selected in step 1), specifically: 2.1) PIT marking is performed on the parents selected in step 1); 2.2) using the microsatellite marker primers of naked carp according to claim 1 to establish a phylogenetic clustering tree for the parents of step 2.1); 2.3) According to the phylogenetic clustering tree constructed in step 2.2), individuals that are not in the same branch of the clustering tree are selected for pairing and breeding to construct different pairing and breeding families; 3) cultivating the paired parents obtained in step 2) to obtain the first generation naked carp; 4) Select backup parents from all the next generation naked carp; 5) The reserve parents selected in step 4) are subjected to kinship identification of the next generation naked carp and parent pairing of the next generation naked carp, specifically: 5.1) PIT marking is performed on the backup parents selected in step 4); 5.2) using the microsatellite marker primer combination of naked carp according to claim 1 to establish a phylogenetic clustering tree for the parents of step 5.1); 5.3) According to the phylogenetic cluster development tree constructed in step 5.2), the first generation of naked carp that are not in the same branch of the cluster development tree are selected for pairing and breeding; 6) culturing the paired first generation naked carp parents obtained in step 5) to obtain second generation naked carp, wherein the second generation naked carp can maintain the excellent growth traits of naked carp; The specific implementation method of the kinship cluster development tree in step 2.2) and step 5.2) is: taking a tissue sample of the naked carp to be identified; extracting DNA of the naked carp sample; performing PCR amplification on the naked carp sample using the 5 groups of naked carp microsatellite marker primer combination double PCR system described in claim 1; performing electrophoresis separation on 10% polyacrylamide gel for the PCR amplification product; statistically calculating the genotype of the PCR amplification product according to the separation result; and establishing a kinship cluster development tree using Mega software according to the genotyping results of each individual.

5. The breeding method for maintaining excellent growth traits of naked carp according to claim 4, characterized in that: The specific implementation method of step 3) is: 3.1) artificially incubating the offspring produced by breeding the paired parents obtained in step 2); 3.2) After hatching, the first generation of naked carp are cultured separately under the same conditions according to different families. During the culture, they are fed with commercial feed three times a day, and the feeding amount is 0.5% of the body weight of the first generation of naked carp.

6. The breeding method for maintaining excellent growth traits of naked carp according to claim 5, characterized in that: The specific implementation method of step 4) is: 4.1) After the first generation of naked carp obtained in step 3) is cultured for one year, the body length and weight of all naked carp in each family are measured, and the first generation of naked carp with excellent traits are selected; the excellent traits are fast growth rate and strong immunity; 4.2) The selected first generation naked carp with excellent traits are cultured to sexual maturity; 4.3) Select the first-generation female and the first-generation male naked carp from each family as backup parents.

7. The breeding method for maintaining excellent growth traits of naked carp according to claim 6, characterized in that: The reaction system of PCR amplification in the kinship identification is 25ul: 10×PCR Buffer 2.5ul, 2.5mmol / L dNTP 1.5ul, 2mmol / L MgCl2 2.5ul, upstream and downstream primers 1ul each, 0.5U / μL Taq enzyme 1ul, DNA template 2ul, ultrapure water 11.5ul; The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing temperature for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min; and storage at 4°C.

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