Crossbreeding method for all-male red cherax quadricarinatus

Abstract

The invention belongs to the technical field of aquatic product breeding, and particularly relates to a cross breeding method for all-male cherax quadricarinatus. The method provided by the invention comprises the following steps: introducing supermale cherax quadricarinatus seeds A, and selecting supermale parent shrimps after breeding; crossbreeding is carried out based on different mating combinations of super-male parent shrimps and pseudo-female parent shrimps, filial generations of different generations are obtained through the modes of orthogonality, reverse crossing, backcrossing and the like, breeding is carried out, and a stably inherited all-male cherax quadricarinatus population is obtained. According to the method provided by the invention, red cherax quadricarinatus with super male and red cherax quadricarinatus with pseudo female are selected for positive and negative cross breeding, and multiple generations of red cherax quadricarinatus with excellent characters are subjected to positive and negative cross selection; and a new red fast-growing variety of all-male cherax quadricarinatus with high growth speed, high-quality meat taste, good disease resistance and strong adaptability is obtained. The new variety has remarkable economic benefits and can be widely applied to shrimp culture and aquatic product markets.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic product breeding, and in particular relates to a hybrid breeding method for all-male red red claw crayfish. Background Art

[0002] Red claw crayfish (Cherax quadricarinatus), native to the streams of tropical areas in northern Australia, looks like a sea lobster and is also known as Australian freshwater lobster. The largest individual weighs about 600g. It is a widely farmed economic shrimp with delicious meat and rich nutrition, and has a large market demand.

[0003] However, existing red claw crayfish varieties have certain deficiencies in meat quality, growth rate, and disease resistance, limiting the economic benefits of their aquaculture. During aquaculture, a red-colored red claw crayfish was discovered, which has more significant commercial traits. Therefore, hybrid breeding of all-male red red claw crayfish is of great significance.

[0004] In 2023, the Institute of Hydrobiology of Zhejiang Academy of Agricultural Sciences, in collaboration with Xiamen University, Yellow Sea Fisheries Research Institute of Chinese Academy of Fishery Sciences, and Qingdao BGI Genomics Institute, took the lead in China in completing the whole genome sequence analysis of red claw crayfish using second-generation sequencing, Pacbio and Hi-C technologies.

[0005] Although many domestic research institutes and enterprises have carried out research on genetic breeding of red claw crayfish, the existing red claw crayfish varieties bred by the existing breeding methods have deficiencies in key economic traits such as meat quality, growth rate and disease resistance. Summary of the Invention

[0006] To address the problem that existing red claw crayfish breeding methods in the prior art lack key economic traits such as meat quality, growth rate, and disease resistance in existing varieties of red claw crayfish, the present invention provides a hybrid breeding method for all-male red red claw crayfish. To achieve this objective, the present invention employs the following technical solutions.

[0007] The invention provides an all-male red red claw crayfish hybrid breeding method, comprising the following steps: selecting super male red red claw crayfish fry as super male broodstock.

[0008] The super male broodstock is orthogonally and / or reciprocally crossed with the pseudo female broodstock, the obtained hybrid offspring is then backcrossed with the pseudo female broodstock, the obtained hybrid offspring is raised as seedlings, and screening is performed based on phenotypic and genetic characteristics to obtain red claw crayfish individuals that meet the requirements of all-male red body color; the screened red claw crayfish individuals that meet the requirements of all-male red body color are raised as seedlings to obtain a stable genetic all-male red red claw crayfish population.

[0009] The present invention provides an all-male red red claw crayfish hybrid breeding method, which comprises the following steps: firstly, super-male red red claw crayfish fry are introduced, and super-male broodstock are selected after cultivation; then, the super-male broodstock are orthogonally and / or reciprocally crossed with pseudo-female broodstock, and the obtained hybrid offspring are backcrossed with the pseudo-female broodstock; the obtained hybrid offspring are raised as seedlings, and are screened according to phenotypic and genetic characteristics to obtain red claw crayfish individuals that meet the all-male red body color; and the screened red claw crayfish individuals that meet the all-male red body color are raised as seedlings to obtain a genetically stable all-male red red claw crayfish population. The present invention utilizes gene recombination through orthogonal reciprocal crosses to break unfavorable linkages, achieves homozygosity of dominant genes through backcrossing, and carries out transgenerational recombination of high-quality traits (super-male superior growth + pseudo-female disease resistance potential + red body color dominance) dispersed in different parents to obtain stably inherited all-male red red claw crayfish. The all-male red red claw crayfish has excellent meat quality, fast growth rate, and good Aeromonas hydrophila disease resistance, and can solve the problem that existing varieties are deficient in key economic traits such as meat quality, growth rate, and disease resistance.

[0010] Preferably, the supermale broodstock include supermale red claw crayfish fry (A) female broodstock and supermale red claw crayfish fry (A) male broodstock. "A" represents first-class or preferred fry, generally referring to high-quality fry with a body length of ≥2 cm, uniform size, strong vitality, no injuries, and a zero disease detection rate. The survival rate is significantly higher than that of ordinary blackhead fry (1 cm to 2 cm).

[0011] The pseudo-female broodstock include pseudo-female male broodstock and pseudo-female female broodstock.

[0012] In some embodiments of the present invention, the supermale broodstock are Australian red red claw crayfish (supermale), Guangdong red red claw crayfish (supermale), and Zhejiang red red claw crayfish (supermale). The pseudo-female broodstock are Guangxi red red claw crayfish (pseudo-female), Guangdong red red claw crayfish (supermale), and Fujian red red claw crayfish (pseudo-female).

[0013] Preferably, the orthogonal cross refers to the mating of the super male red claw crayfish fry male broodstock with the pseudo female female broodstock, and the hybrid offspring obtained is the orthogonal B1 + Orthogonal B1 + The offspring are the offspring of the super male red red claw crayfish fry male broodstock and the pseudo female broodstock.

[0014] The backcross refers to the mating of the super male red claw crayfish female parent shrimp with the pseudo female male parent shrimp, and the hybrid offspring obtained is the backcross B1 - . Backcross B1 -为 The offspring are the offspring of the super male red red claw crayfish fry female broodstock and the pseudo female male broodstock.

[0015] Preferably, the backcrossing comprises an orthogonal backcrossing.

[0016] The orthogonal backcrossing includes the orthogonal B1 + The hybrid offspring obtained by mating with the pseudo-female male broodstock is B1 + Orthocross progeny B2 ++ ; and / or the backcross B1 - The hybrid offspring obtained by mating with the pseudo-female male broodstock is B1 - Orthocross progeny B2 -+ .

[0017] Preferably, the B1 + Orthocross progeny B2 ++ and B1 - Orthocross progeny B2 -+ The seedlings were raised separately to obtain red claw crayfish individuals with all-male red body color.

[0018] Preferably, the backcrossing further comprises reverse backcrossing.

[0019] The reverse backcross includes the reverse cross B1 - The hybrid offspring obtained by mating with the pseudo-female female shrimp is B1 + Backcross progeny B2 +- ; and / or the backcross B1 - The hybrid offspring obtained by mating with the pseudo-female female shrimp is B1 - Backcross progeny B2 -- .

[0020] Preferably, the B1 - Backcross progeny B2 -- and B1 - Backcross progeny B2 -- The seedlings were raised separately to obtain red claw crayfish individuals with all-male red body color.

[0021] Preferably, shrimps with strong physique, complete appendages and no diseases are selected from the all-male red claw crayfish individuals for seed breeding and cultivation to obtain a stable genetic all-male red claw crayfish population.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for hybrid breeding of all-male red red claw crayfish. The all-male red red claw crayfish hybrid breeding method provided by the present invention first selects super-male red red claw crayfish fry, and after cultivation, selects super-male broodstock; then, orthogonal and / or reciprocal crosses are performed on the super-male broodstock with pseudo-female broodstock, and the resulting hybrid offspring are backcrossed with the pseudo-female broodstock; the resulting hybrid offspring are raised as seedlings, and are screened based on phenotypic and genetic characteristics to obtain red claw crayfish individuals that meet the all-male red body color; the screened red claw crayfish individuals that meet the all-male red body color are raised as seedlings to obtain a genetically stable all-male red red claw crayfish population. The present invention utilizes gene recombination through orthogonal reciprocal crosses to break unfavorable linkages, achieves homozygosity of dominant genes through backcrossing, and carries out transgenerational recombination of high-quality traits (super-male superior growth + pseudo-female disease resistance potential + red body color dominance) dispersed in different parents to obtain stably inherited all-male red red claw crayfish. The all-male red red claw crayfish has excellent meat quality, fast growth rate, and good Aeromonas hydrophila disease resistance, and can solve the problem that existing varieties are deficient in key economic traits such as meat quality, growth rate, and disease resistance.

[0023] 2. B1 bred by the present invention + Orthogonal backcross B2 ++ 、B1 + Backcross B2 +- 、B1 - Backcross B2 -- and B1 - Orthogonal backcross B2 -+ The survival rate and fertilization rate of the four types of seedlings were all good, and the moisture, crude ash, crude protein, crude fat content and the composition of amino acids and fatty acids were significantly higher than those of ordinary red claw crayfish. This shows that the commercial traits of the local cultured population of red claw crayfish can be greatly improved by introducing super male populations and pseudo female populations for hybridization and breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of orthogonal B1+ breeding in the present invention.

[0025] Figure 2 Schematic diagram of reciprocal cross B1-breeding in the present invention.

[0026] Figure 3 This is a schematic diagram of B1+ backcross B2++ breeding in the present invention.

[0027] Figure 4 This is a schematic diagram of B1+ reverse backcross B2+- breeding in the present invention.

[0028] Figure 5 This is a schematic diagram of B1-backcross B2-breeding in the present invention.

[0029] Figure 6This is a schematic diagram of B1-backcross B2-+ breeding in the present invention. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] Example 1: Hybrid breeding method of Australian red red claw crayfish (super male) and Guangxi red red claw crayfish (pseudo female) Screening of broodstock: Screening of broodstock is carried out by the following method, including the determination of economic traits and nutritional quality.

[0032] 1) Methods for determining economic traits: (1) Every 10 days, weigh and measure the body length of the marked broodstock and record the data. After the culture is completed, calculate the average daily weight gain, specific growth rate, and survival rate.

[0033] The calculation formula for average daily weight gain is as follows: Average daily weight gain = (final weight - initial weight) / number of days of breeding; In the above formula, the English name of average daily weight gain is ADG, and the unit is g / d; final weight refers to the weight of shrimp at the end of culture; initial weight refers to the weight of shrimp before culture begins; The specific growth rate is calculated as follows: Specific growth rate = [ln (final weight) - ln (initial weight)] / number of culture days × 100%; In the above formula, the English name of specific growth rate is SGR, and the unit is % / d; The survival rate is calculated as follows: Survival rate = number of surviving shrimp / initial number of shrimp × 100%; In the above formula, the English name of survival rate is SR, and the unit is %; the number of surviving shrimps refers to the number of shrimps at the end of farming; the initial number of shrimps refers to the number of shrimps at the beginning of farming.

[0034] (2) Ten pairs of sexually mature parents, each from the Zhejiang population, were selected and housed individually in a 1 m × 1 m × 0.6 m breeding pond. The water temperature was controlled at (28 ± 1)°C, and the photoperiod was 12 L:12 D. The spawning time and egg mass weight of the female shrimp were recorded, and the egg diameter was observed using a microscope (10× objective lens) (the average value of 20 eggs was measured). After the larvae hatched, the hatching rate of the larvae was calculated.

[0035] The calculation formula for hatching rate is as follows: Hatching rate = number of hatched larvae / total number of eggs × 100%; In the above formula, the English name of hatching rate is HR, and the unit is %; the number of hatched larvae refers to the number of surviving eggs carried by female shrimp that successfully break away from the egg membrane and develop into independent larvae (usually referring to the zoea or juvenile shrimp stage) after hatching; the total number of eggs refers to the total number of mature eggs carried by the female shrimp in one reproductive cycle (that is, the number of all eggs attached to the female shrimp's abdominal limbs from spawning to hatching).

[0036] The Zhejiang population refers to the red claw crayfish population introduced from Australia that was successfully domesticated and stably inherited in the Zhejiang region, and its origin is Australia.

[0037] Sexually mature parents refer to male and female individuals that have reached physiological maturity and are capable of reproduction, and they originate from Australia.

[0038] (3) Artificial infection experiments were conducted using Aeromonas hydrophila. The activated strain was cultured to the logarithmic growth phase and the bacterial solution concentration was adjusted to 1×10 6 CFU / mL. Thirty shrimp from each of the two populations were randomly selected and intraperitoneally injected with 0.1 mL of bacterial solution. A control group was injected with an equal volume of saline. Mortality was observed and recorded over a 7-day period. Cumulative mortality was calculated, and survival curves were plotted.

[0039] The calculation formula for the cumulative mortality rate is as follows: Cumulative mortality rate = (number of individuals who died during the experiment / total number of individuals in the initial experiment) × 100%; In the above formula, the English name of the cumulative mortality rate is CM, and the unit is %.

[0040] The strain used in the above method refers to Aeromonas hydrophila AH10, numbered CCTCC AB 2014155, purchased from China Center for Type Culture Collection.

[0041] 2) Methods for determining nutritional quality: (1) Moisture content was determined by direct drying at 105°C (GB 5009.3-2016); crude protein content was determined by the Kjeldahl method (GB 5009.5-2016); crude fat content was determined by the Soxhlet extraction method (GB 5009.6-2016); and ash content was determined by the 550°C incineration method (GB 5009.4-2016).

[0042] (2) Amino acid determination: Weigh 0.2 g of dried sample, add 6 mol / L hydrochloric acid, and hydrolyze at 110°C for 24 h. After filtration and concentration, the hydrolyzate was analyzed by high-performance liquid chromatography (HPLC) (Agilent 1260) using the pre-column derivatization method with o-phthalaldehyde (OPA) and 9-fluorenyl chloroformate (FMOC) to determine the contents of 17 amino acids (for the method, see “Determination of amino acid content in silver carp, bighead carp, and grass carp muscle by high-performance liquid chromatography” by Li Yan, Xie Yong, and Yang Fahui, published in the Hubei Journal of Agricultural Sciences in 2018).

[0043] (3) Fatty acid determination: Fat was extracted using the chloroform-methanol method, methylated, and analyzed using a gas chromatography-mass spectrometer (GC-MS, Thermo Scientific ISQ 7610). The column used was a DB-23 (30 m × 0.25 mm × 0.25 μm) column. The temperature program was as follows: initial temperature 100°C, increasing to 220°C at a rate of 5°C / min, and maintaining for 15 min.

[0044] By comparing the above economic traits and nutritional quality test results, two excellent local populations of Australian red claw crayfish and Guangxi red claw crayfish were found.

[0045] The shrimp species of the above two excellent local populations were artificially propagated separately. The specific steps were as follows: (1) Screening criteria for broodstock: From the excellent populations of Australian red claw crayfish and Guangxi red claw crayfish, broodstock with strong physique, complete appendages and no diseases were selected. Among them, the weight of female shrimp was required to reach 90g±10g and the body length was 11cm±1cm; the weight of male shrimp was required to reach 110g±10g and the body length was 13cm±1cm. Sexual maturity was determined by observing the color of the gonads (the ovaries of female shrimp were brown, and the testes of male shrimp were milky white). 100 pairs of broodstock were selected from each population, for a total of 200 pairs. Among them, the Australian red claw crayfish (super male) was purchased from Zhejiang Haining Honghai Breeding Co., Ltd.; and the Guangxi red claw crayfish (pseudo female) was sourced from the Guangxi Academy of Fisheries Sciences.

[0046] (2) Induced spawning: After 4 months of broodstock cultivation, oxytocin was artificially injected to induce spawning. Oxytocin is a luteinizing hormone-releasing hormone analogue (LHRH-A2). The injection dose is 0.5 μg / g body weight for females and 0.3 μg / g body weight for males. The injection site is the ventral surface where the cephalothorax and abdomen meet. The injected broodstock are placed in a 1:1 ratio of male to female in spawning ponds with a size of 2m×1.5m×0.8m. One pair of broodstock is placed in each spawning pond, and a total of 200 spawning ponds are set up.

[0047] After the shrimp larvae are hatched, they are cultured in special ponds. The specific steps are as follows: select a rectangular pond with an area of ​​4 mu, a depth of 2m, a slope ratio of 1:2.5, a flat bottom, and a silt thickness of no more than 10cm. Use 10 ponds for each population, for a total of 20 ponds. Plant aquatic plants around and in the middle of the pond, with the planting area accounting for 35% of the total area of ​​the pond, to provide a place for the experimental red claw crayfish to live, molt, and avoid enemies. 1,000 shrimp fry with a body length of 0.6cm are put into each pond, and the water temperature difference does not exceed 2°C when they are put in. Shrimp fry of the same size are slowly put into the upwind side of the pond, and the shrimp fry that have adapted to the environment are allowed to swim into the pond on their own.

[0048] After sex differentiation, manual selection is performed. The specific steps are as follows: After 56 days of culture, when the shrimp reach 7 cm in length, the first sexing is performed. Visual observation reveals a pair of male appendages on the inner side of the base of the fifth leg of male shrimp, while female shrimp have a pair of genital pores on the inner side of the base of the third leg. Finally, 200 female shrimp are removed from the pond, leaving 700 super-male shrimp.

[0049] 24 hours after the mating of pseudo-male and pseudo-female shrimps, 800 red pseudo-female shrimps carrying eggs were taken out from the breeding pond, and then the eggs of the red pseudo-female shrimps carrying eggs were removed by an efficient and non-destructive egg removal method, specifically as follows: (1) Selecting egg-carrying shrimps: Select egg-carrying female shrimps that are disease-free, uninjured, and have good vitality, and raise them in cages for later use.

[0050] (2) Cleaning the egg removal pool: The egg removal pool is a cement pool 2 meters long, 1 meter wide, and 1 meter high. The bottom of the pool is equipped with 25 evenly distributed aeration heads, and a water outlet is opened at the lowest point in the center of the pool. Before use, clean the egg removal pool with tap water and rinse it once or several times with deionized water.

[0051] (3) Prepare egg removal solution: put 1000 kg of deionized water at 28°C into the egg removal tank, first add 8 kg of chemically pure sodium chloride and stir until it is completely dissolved; then add 4 kg of protease and stir until it is completely dissolved.

[0052] (4) Despawning: Open the aerator and aerate for 5 minutes, then add 57 kg of brooding shrimp. The water temperature during the despawning process is kept at 28°C, and continuous aeration is maintained at 70 watts per ton of water until all eggs fall off the abdomen of the brooding shrimp.

[0053] (5) Collection: After the eggs are removed, the pseudo-female shrimps are picked out with a net and placed in a pond for breeding. During the transfer process, the time the female shrimps are out of water should be shortened as much as possible to reduce damage; the free eggs that fall off are filtered and collected with a 100-mesh net.

[0054] The artificial propagation and shrimp seed cultivation of red claw crayfish were carried out by hybridizing the pseudo-female red claw crayfish from Guangxi and the super male red claw crayfish from Australia to obtain high-quality shrimp seedlings. The specific method is as follows:

[0055] One thousand pseudo-female red claw crayfish from Guangxi and super-male red claw crayfish from Australia were selected, and the super-male broodstock of red claw crayfish from Australia was mated with the pseudo-female broodstock of red claw crayfish from Guangxi to obtain orthogonal B1. + , Guangxi red red claw crayfish pseudo-male and female broodstock and Australian red red claw crayfish super-male and female broodstock to obtain backcross B1 - ; Take orthogonal B1 + Backcross with Guangxi pseudo-male and female shrimp, backcross B1 - Backcrossing with Guangxi pseudo-female shrimp resulted in B1 + Orthocross progeny B2 ++ 、B1+backcross progeny B2 +- 、B1 - Backcross progeny B2 -- and B1 - Orthocross progeny B2 -+ ; For B1 + Orthocross progeny B2 ++ 、B1 + Backcross progeny B2 +- 、B1-backcross progeny B2 -- and B1 - Orthocross progeny B2 -+ The broodstock were reared separately to obtain all-male red-colored red claw crayfish. From these all-male red claw crayfish, broodstock with robust physique, intact appendages, and no disease were selected. Females were required to weigh 80g and be 12cm long; males 110g and 13cm long. Sexual maturity was determined by observing the color of the gonads (ovaries in females were brown, while testes in males were milky white). 200 pairs of broodstock were selected from each population, for a total of 400 pairs. After four months of rearing, the selected broodstock were artificially injected with oxytocin, a luteinizing hormone-releasing hormone analog (LHRH-A2), at a dose of 0.5μg / g body weight for females and 0.3μg / g body weight for males. The injection site was the ventral surface where the cephalothorax and abdomen meet. The injected broodstock were placed in a 1:1 ratio of male to female in spawning ponds measuring 2m x 1.5m x 0.8m, with one pair of broodstock placed in each pond. A total of 200 spawning ponds were set up. The resulting broodstock produced 52,601 shrimp fry with rapid growth, high-quality meat flavor, good disease resistance, and strong adaptability.

[0056] Example 2: Hybrid breeding method of Guangxi red red claw crayfish (pseudo-female) and Guangdong red red claw crayfish (super male) 1) Method for determining economic traits (same as in Example 1).

[0057] (1) Method for determining nutritional quality (same as in Example 1).

[0058] (2) Amino acid determination (same as Example 1).

[0059] (3) Fatty acid determination (same as Example 1).

[0060] By comparing the above economic traits and nutritional quality test results, two excellent local populations of Guangxi red claw crayfish and Guangdong red claw crayfish were found.

[0061] The Guangxi red pseudo-female red claw crayfish and the Guangdong red super-male red claw crayfish were used as broodstock for cross breeding. One thousand Guangxi red pseudo-female red claw crayfish and Guangdong red super-male red claw crayfish were selected, and the Guangdong red red claw crayfish super-male broodstock were mated with the Guangxi red red claw crayfish pseudo-female broodstock to obtain orthogonal B1 + , Guangxi red red claw crayfish pseudo-male and female broodstock and Guangdong red red claw crayfish super-male and female broodstock to obtain backcross B1 - Among them, the source of Guangdong red claw crayfish (super male) is Guangdong Aoda Investment Development Co., Ltd.

[0062] Take orthogonal B1 + Backcross with Guangxi pseudo-male and female shrimp, backcross B1-backcross with Guangxi pseudo-female shrimp to obtain B1 + Orthocross progeny B2 ++ 、B1+backcross progeny B2 +- 、B1-backcross progeny B2 -- and B1 - Orthocross progeny B2 -+ .

[0063] For B1 + Orthocross progeny B2 ++ 、B1+backcross progeny B2 +- 、B1-backcross progeny B2 -- and B1 - Orthocross progeny B2 -+ The seedlings were raised separately to obtain red claw crayfish individuals with all-male red body color.

[0064] The population expansion and picking methods are the same as those in Example 1, and shrimp fry with high-speed growth, high-quality meat flavor, good disease resistance and strong adaptability can be obtained.

[0065] High-quality shrimp fry were obtained by hybridizing Guangxi red pseudo-female red claw crayfish with Guangdong red super male red claw crayfish for artificial breeding and shrimp fry cultivation.

[0066] Example 3: Hybrid breeding method of Fujian red red claw crayfish (pseudo-female) and Zhejiang red red claw crayfish (super male) 1) Method for determining economic traits (same as in Example 1).

[0067] (1) Method for determining nutritional quality (same as in Example 1).

[0068] (2) Amino acid determination (same as Example 1).

[0069] (3) Fatty acid determination (same as Example 1).

[0070] By comparing the above economic traits and nutritional quality test results, two excellent local populations of Fujian red claw crayfish and Zhejiang red claw crayfish were found.

[0071] Crossbreeding was carried out using Fujian red pseudo-female red claw crayfish and Zhejiang red super-male red claw crayfish as broodstock. One thousand Fujian red pseudo-female red claw crayfish and Zhejiang red super-male red claw crayfish were selected, and the Zhejiang red red claw crayfish super-male broodstock were mated with Fujian red red claw crayfish pseudo-female broodstock to obtain orthogonal B1. + , the pseudo-male and female broodstock of Fujian red red claw crayfish and the super-male and female broodstock of Zhejiang red red claw crayfish were reciprocally crossed to obtain B1 - The Fujian red claw crayfish (pseudo-female) was sourced from Liming Aquatic Products Breeding Co., Ltd. in Luoyuan County, Fujian Province, and the Zhejiang red claw crayfish (super-male) was sourced from Zhejiang Jinlanlong Agricultural Technology Co., Ltd.

[0072] Take orthogonal B1 + Backcross with Fujian pseudo-male and female shrimp, backcross B1-backcross with Fujian pseudo-female shrimp to obtain B1 + Orthocross progeny B2 ++ 、B1 + Backcross progeny B2 +- 、B1-backcross progeny B2 -- and B1 - Orthocross progeny B2 -+ .

[0073] For B1 + Orthocross progeny B2 ++ 、B1+backcross progeny B2 +- 、B1-backcross progeny B2 -- and B1 - Orthocross progeny B2 -+ The seedlings were raised separately to obtain red claw crayfish individuals with all-male red body color.

[0074] The population expansion and picking methods are the same as those in Example 1, and shrimp fry with high-speed growth, high-quality meat flavor, good disease resistance and strong adaptability can be obtained.

[0075] The artificial propagation and fry cultivation of red claw crayfish were carried out by hybridization of Fujian red pseudo-female red claw crayfish and Zhejiang red super-male red claw crayfish to obtain high-quality fry.

[0076] Comparative Example 1: A primer pair for breeding large-sized and fast-growing red claw crayfish strains and corresponding probes, kits, methods and applications Method steps: Breeding conditions: density 30 / m³, water temperature 26°C, dissolved oxygen 6 mg / L, feeding twice a day.

[0077] Parental mating: Red claw crayfish (weighing ≥ 100g) were selected from the Zhejiang and Guangdong populations, with a male-to-female ratio of 2:1. After natural mating, spawning females were individually reared to establish full-sib families. The red claw crayfish from the Zhejiang population were sourced from Zhejiang Jinlanlong Agricultural Technology Co., Ltd. The red claw crayfish from the Guangdong population were sourced from Guangdong Aoda Investment Development Co., Ltd.

[0078] Delayed screening: After 4 months of breeding, select families with a survival rate ≥ 90% and a weight gain multiple ≥ 50 times, extract DNA from 5 shrimp tails, and use primer pairs and probes to detect and screen qualified families.

[0079] Inbreeding: Continuous mating for two generations within a qualified family (F1→F2).

[0080] Hybrid propagation: The F2 generation was mated with red claw crayfish (weight ≥ 100g) from Fujian population to obtain the F3 generation, and the F generation was self-pollinated for two consecutive generations to obtain shrimp fry.

[0081] Comparative Example 2: A method for constructing a red claw crayfish family using in vitro hatching technology Method steps: Parent screening and introduction: 10 inbred groups were selected from Hainan South Breeding Base, Guangdong Aoda Investment Development Co., Ltd., Zhejiang Jinlanlong Agricultural Technology Co., Ltd. and other regions. After genetic diversity testing and virus PCR testing, 3 groups with long genetic distance and no virus were selected (female shrimp ≥50g, male shrimp ≥60g) and isolated for breeding.

[0082] Intensive cultivation: Cultivate in a greenhouse with controlled temperature, with the water temperature rising from 20°C to 28°C (1°C per day), then slowly cooling down to 20°C, and returning to 29°C before mating; feed containing Clostridium butyricum, supplemented with carrots, hairtail, etc., and add Bacillus subtilis every week to regulate water quality.

[0083] Family construction: 1 male shrimp and 5 female shrimp were mated, and orthogonal crosses, reciprocal crosses and self-crosses were performed to establish 600 mating groups (570 orthogonal and reciprocal crosses + 30 self-crosses). The shrimp were reared in 300-liter blue barrels and set up shrimp houses for shelter.

[0084] In vitro hatching: 40 days after mating, remove shrimp eggs at the five / seven pairs of appendages and compound eye pigment stages, disinfect them with 3000PPM formaldehyde for 15 minutes, and place them in a shaking incubator (water temperature 29℃, dissolved oxygen ≥8mg / L). Clean the dirt and disinfect the unhatched eggs daily, and scan and count them with a precision camera.

[0085] Standardized thickening: Blackhead seedlings are transferred to thickening barrels (120 fish / barrel), fed with Artemia, cladopods and mixed feed, and thickened to 5cm in 60 days. Bacillus is supplemented weekly to prevent Vibrio.

[0086] Fluorescent labeling and same-pond culture: Seven fluorescent dyes were injected into the antennae and abdomen of shrimp fry (one color combination for each family), and families with few or small fry were eliminated; the shrimp were cultured in separate ponds in May, fed three times a day, and the water quality was monitored.

[0087] Phenotypic determination and cyclic mating: In October, the broodstock are caught again, and the weight, body length and other data are measured. The breeding value is calculated using the BLUP method, and 200 high-quality families are selected and re-mated to enter the next breeding cycle.

[0088] In order to illustrate the effect of the hybrid breeding method of an all-male red red claw crayfish provided by the present invention, we take the above-mentioned Example 1 and Comparative Examples 1 to Comparative Examples 2 as examples, and test the shrimp fry of Comparative Example 1 with high survival rate, fast growth rate and stable traits and the shrimp fry of Comparative Example 2 with fast growth rate and high survival rate, which were harvested after 60 days of culture. The results are shown in Tables 1 to 4.

[0089] Table 1 Growth performance Table 2 Disease resistance (Aeromonas hydrophila infection) Table 3 Amino acid composition (mg / 100g) Table 4 Fatty acid composition (% of total fatty acids) It can be seen from Tables 1 to 4 that the all-male red crayfish cultivated in Example 1 are superior to those in Comparative Examples 1 and 2 in terms of growth performance, disease resistance and nutritional components.

[0090] The final weight of Example 1 (32.5±2.1g) was significantly higher than that of Comparative Example 1 (25.8±1.8g) and Comparative Example 2 (28.7±2.0g), and the average daily weight gain (0.356g / d) and specific growth rate (2.89% / d) were also better than those of the two comparative examples, indicating that the technical solution of the present invention can effectively improve the growth rate of red claw crayfish, shorten the breeding cycle, and solve the problem of poor growth performance in the prior art.

[0091] In the Aeromonas hydrophila infection test, the cumulative mortality rate of Example 1 (15.0±2.0%) was significantly lower than that of Comparative Example 1 (32.0±3.5%) and Comparative Example 2 (22.0±2.8%), and the median lethal time (96.5h) was longer, indicating that the red claw crayfish cultivated by the technical solution of the present invention has stronger disease resistance, can reduce losses caused by diseases, and overcome the defect of insufficient disease resistance in the prior art.

[0092] The total amount of essential amino acids (3820±120 mg / 100 g), the total amount of umami amino acids (4150±105 mg / 100 g) and the content of key amino acids (methionine, lysine) in Example 1 were all higher than those in the comparative example. At the same time, the proportion of unsaturated fatty acids such as EPA and DHA (1.8±0.2% and 3.5±0.3%) was better, indicating that the technical solution of the present invention can improve the nutritional components and edible value of red claw crayfish and solve the problem of uneven nutritional quality in the prior art.

[0093] From the above results, it can be seen that the technical solution of the present invention can optimize the growth performance of red claw crayfish, enhance disease resistance and improve nutritional quality, and effectively solve the core technical problems existing in the existing red claw crayfish breeding.

[0094] In summary, the all-male red red claw crayfish hybrid breeding method provided by the present invention significantly breaks through the bottlenecks of existing breeding methods in efficiency, survival rate and quality through targeted hybridization and efficient egg removal technology, and has significant technological progress and industrial application value.

[0095] It should be noted that the above examples only illustrate the all-male red claw crayfish hybrid breeding technology established by the present invention and the selected all-male red claw crayfish hybrid combinations, but the present invention is not limited thereto. It is worth noting that the new all-male red claw crayfish intraspecific hybrid strain obtained through the above-mentioned technical route and the shrimp fry production using the above-mentioned technical route are all within the scope of protection claimed by the present invention and do not depart from the scope of protection of the present invention and the appended claims. Other specific embodiments will not be listed here one by one.

[0096] The present invention mainly cultivates specialized strains with prominent main selected traits and excellent comprehensive performance, realizes comprehensive improvement of multiple traits of red claw crayfish through hybridization, cultivates new red, fast-growing varieties of all-male red claw crayfish, and promotes large-scale breeding.

[0097] When the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0098] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for hybrid breeding of all-male red red claw crayfish, characterized in that: The steps include: selecting super male red red claw crayfish fry as super male broodstock; The super male broodstock is orthogonally and / or reciprocally crossed with the pseudo female broodstock, the obtained hybrid offspring is then backcrossed with the pseudo female broodstock, the obtained hybrid offspring is raised as seedlings, and screening is performed based on phenotypic and genetic characteristics to obtain red claw crayfish individuals that meet the requirements of all-male red body color; the screened red claw crayfish individuals that meet the requirements of all-male red body color are raised as seedlings to obtain a stable genetic all-male red red claw crayfish population.

2. The all-male red red claw crayfish hybrid breeding method according to claim 1, characterized in that: The supermale broodstock include supermale female broodstock of red red claw crayfish fry and supermale male broodstock of red red claw crayfish fry; The pseudo-female broodstock include pseudo-female male broodstock and pseudo-female female broodstock.

3. The all-male red red claw crayfish hybrid breeding method according to claim 2, characterized in that: The orthogonal cross refers to the mating of the super male red claw crayfish fry male broodstock with the pseudo female female broodstock, and the hybrid offspring obtained is the orthogonal B1 + ; The backcross refers to the mating of the super male red claw crayfish female parent shrimp with the pseudo female male parent shrimp, and the hybrid offspring obtained is the backcross B1 - .

4. The all-male red red claw crayfish hybrid breeding method according to claim 3, characterized in that: The backcrossing includes orthogonal backcrossing; The orthogonal backcrossing includes the orthogonal B1 + The hybrid offspring obtained by mating with the pseudo-female male broodstock is B1 + Orthocross progeny B2 ++ ; and / or the backcross B1 - The hybrid offspring obtained by mating with the pseudo-female male broodstock is B1 - Orthocross progeny B2 -+ .

5. The all-male red red claw crayfish hybrid breeding method according to claim 4, characterized in that: Regarding B1 + Orthocross progeny B2 ++ and B1 - Orthocross progeny B2 -+ The seedlings were raised separately to obtain red claw crayfish individuals with all-male red body color.

6. The all-male red red claw crayfish hybrid breeding method according to claim 4, characterized in that: The backcrossing also includes reverse backcrossing; The reverse backcross includes the reverse cross B1 - The hybrid offspring obtained by mating with the pseudo-female female shrimp is B1 + Backcross progeny B2 +- ; and / or the backcross B1 - The hybrid offspring obtained by mating with the pseudo-female female shrimp is B1 - Backcross progeny B2 -- .

7. The all-male red red claw crayfish hybrid breeding method according to claim 6, characterized in that: Regarding B1 - Backcross progeny B2 -- and B1 - Backcross progeny B2 -- The seedlings were raised separately to obtain red claw crayfish individuals with all-male red body color.

8. The all-male red red claw crayfish hybrid breeding method according to any one of claims 5 or 7, characterized in that: From the all-male red claw crayfish individuals, shrimps with strong physique, complete appendages and no diseases are selected for seed breeding and cultivation to obtain a stable genetic all-male red claw crayfish population.

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