Method for creating fast-growing gynogenetic grass carp

Abstract

The application belongs to the technical field of fish variety improvement, and discloses a method for creating gynogenetic grass carp with rapid growth. The method comprises the following steps: screening sexually mature male mirror carp and sexually mature female grass carp with obvious growth advantages and stress resistance, collecting sperm of the mirror carp, diluting the sperm with Hank's solution, inactivating the sperm by ultraviolet irradiation, mixing the obtained inactivated sperm with middle-stage eggs of the grass carp, and then performing cold shock doubling treatment, and adding normal temperature water in batches for hatching to obtain gynogenetic grass carp. The gynogenetic grass carp induced by the mirror carp sperm performs excellently in growth speed and disease resistance, which provides a new method and idea for inheritance improvement of the grass carp, provides more possibilities for screening of excellent varieties, and brings greater economic benefits to the grass carp breeding industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fish variety improvement, and particularly relates to a method for creating a gynogenesis grass carp variety with rapid growth characteristics by using mirror carp (Cyprinus carpio var. Cyprinus carpio var. specularis Ctenopharyngodon idella ) sperm to induce gynogenesis of grass carp. BACKGROUND

[0002] Grass carp (Ctenopharyngodon idellus) is one of the important freshwater fish in China, which has the advantages of herbivorous, fast growth, delicious meat and the like, but the overwintering syndrome causes great economic losses to the grass carp breeding industry, and there is no effective treatment method at present, resulting in low survival rate of grass carp and serious restriction on the development of the grass carp breeding industry and the like. Figure 1 In order to improve the quality of grass carp germplasm and increase the survival rate of grass carp overwintering, it is an effective way to cultivate a new excellent grass carp variety with rapid growth characteristics by using gynogenesis technology.

[0003] Gynogenesis is a kind of genetic breeding technology using heterologous sperm to stimulate the development of eggs, but not transmitting paternal genetic material, which can produce all-female offspring and improve breeding efficiency and quality. These heterologous sperm do not participate in heredity, but may introduce or create some excellent traits through integration or recombination of chromosomes or DNA fragments. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background technology, and to provide a method for creating gynogenesis grass carp with rapid growth characteristics, which can obviously improve the overwintering survival rate of gynogenesis grass carp pure line population.

[0005] To solve the above technical problems, the technical solution provided by the present application is as follows:

[0006] A method for creating gynogenesis grass carp with rapid growth, comprising the following steps:

[0007] (1) selecting sexually mature male mirror carp and sexually mature female grass carp with obvious growth advantages and stress resistance;

[0008] (2) collecting mirror carp sperm and diluting with Hank's solution, and inactivating the sperm by ultraviolet irradiation;

[0009] (3) mixing the obtained inactivated sperm with middle section eggs of grass carp, then doubling the treatment by cold shock, and then adding normal temperature water in batches for hatching to obtain gynogenesis grass carp.

[0010] Mirror carp is an excellent carp breed through artificial selection, which has the characteristics of fast growth, strong resistance, strong cold resistance and high disease resistance. Using mirror carp sperm as the stimulus source of grass carp gynogenesis, the excellent traits of mirror carp such as fast growth, low temperature tolerance and low oxygen tolerance can be transferred or created to gynogenetic grass carp, so as to obtain gynogenetic grass carp with fast growth and stress resistance and cultivate excellent new grass carp breeds.

[0011] Preferably, in step (1), to obtain a gynogenetic grass carp with fast growth, healthy mirror carps with high specific growth rate and good sperm motility are selected as the male parent, and healthy grass carps with high specific growth rate are selected as the female parent; specifically, by measuring the weight change of mirror carps of the same age, the growth rate is used as a direct standard to judge whether the mirror carps have growth advantage, and healthy sexually mature male mirror carp individuals with obvious growth advantage and stress resistance are screened from the mirror carp population; by measuring the weight change of grass carps of the same age, the growth rate is used as a direct standard to judge whether the grass carps have growth advantage, and healthy sexually mature female grass carp individuals with obvious growth advantage are screened from the grass carp population.

[0012] Preferably, the specific growth rate SGR is used to judge the speed of growth, and the calculation formula is , wherein WB is the weight (g) at the starting time, WE is the weight (g) at the ending time, and t is the length of time (days).

[0013] Preferably, in step (2), the microscope is used to detect the sperm motility of mirror carps, and the sperm of mirror carps with long survival time and good motility is selected, diluted with 4-4 times the volume of Hank's solution, mixed uniformly, and then spread on the culture dish according to the standard of 0.1-0.2 mm thick. The culture dish containing the sperm is placed on the shaking table with an ice bag, and the sperm is inactivated under dark environment by using two 15-watt ultraviolet lamps, the perpendicular distance between the sperm and the ultraviolet lamp is 30-35 cm, and the irradiation time is 43-52 min. The above method ensures uniform irradiation of sperm and reduces the rate of sperm inactivation, reduces the over-inactivation of sperm caused by uneven sampling or too fast sperm inactivation (generally, the mirror test uses the sperm tail to swing in water droplets, which is 70-80% weaker than before inactivation), so as to cause the inactivation of grass carp eggs.

[0014] Preferably, in step (2), the Hank's solution comprises the following components in mass fraction: NaCl 8-8.2 parts, KCl 0.4-0.45 parts, CaCl2 0.14-0.15 parts, MgSO4·7H2O 0.2-0.25 parts, Na2HPO4·H2O 0.06-0.065 parts, KH2PO4 0.06-0.065 parts, NaHCO3 0.35-0.36 parts, C6H 12O61.0-1.5 parts, MgCl2·6H2O 0.1-0.15 parts and water 1000 parts.

[0015] Preferably, the rotation speed of the shaker is 80-120 r / min, and the sperm is kept at 4-6 ℃.

[0016] Preferably, the inactivation standard of the sperm is that the microscope examination shows that 20-30% of the sperm has normal activity after being activated by water.

[0017] Preferably, in step (3), the obtained inactivated sperm is mixed with the middle-stage ovum of grass carp, normal temperature water is added and the mixture is fully stirred with a feather, the fertilized ovum after the cold treatment is transferred into 24-26 ℃ warm water for treatment for 4-6 min, and the double-treated fertilized ovum is obtained.

[0018] In the natural or artificial breeding process of grass carp, the spawning behavior of female fish can be divided into multiple stages (such as early stage, middle stage and late stage). The ovum discharged in the middle stage is specifically the ovum discharged in the peak spawning period (i.e. the middle stage). The ovum in this stage usually has higher maturity and fertilization potential, and is a key part of successful breeding.

[0019] Preferably, the double-treated fertilized ovum is added into normal temperature water in batches, and when the water temperature is consistent with the water temperature in the hatching device, the hatching device is transferred into the hatching device for normal hatching, and the obtained fry after hatching is transferred to a pre-fertilized cement pool for early feeding, so that the gynogenetic grass carp is obtained.

[0020] Preferably, the hatching is flow hatching, and the water flow is suitable to roll up the fertilized ovum and tumble, and the water temperature of the hatching barrel for hatching is 22-26 ℃.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] 1. The mirror carp selected in the present application is an excellent carp variety selected by artificial breeding, which has the characteristics of fast growth, strong stress resistance, low temperature resistance, high disease resistance and the like, and the parent generation is screened by using specific growth rate, the gynogenesis process is optimized and innovated, and the gynogenetic grass carp with fast growth and strong stress resistance is successfully developed.

[0023] 2. It is found that the specific growth rate (one and a half years) of the gynogenetic grass carp induced by mirror carp sperm is 0.104 and 0.034 higher than that of the gynogenetic grass carp induced by improved red carp sperm and the gynogenetic grass carp induced by mirror carp sperm respectively, and 0.114 higher than that of ordinary grass carp, and the survival rate is obviously better than that of the gynogenetic grass carp induced by improved red carp sperm and ordinary grass carp, which provides a new method and idea for the genetic improvement of grass carp, provides more possibilities for screening excellent varieties, and brings greater economic benefits to the grass carp breeding industry. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0025] Figure 2 is a flow chart of mirror carp sperm inducing gynogenesis;

[0026] Figure 3 is a flow chart of red crucian carp sperm inducing gynogenesis;

[0027] Figure 4 is a flow chart of mirror carp sperm inducing gynogenesis;

[0028] Figure 5 is a comparison chart of the external appearance of three kinds of gynogenetic grass carps and common grass carps;

[0029] Figure 6 is a chart of grass carp kidney cell mitotic metaphase chromosomes;

[0030] Figure 7 is a result of grass carp male sex-specific molecular marker;

[0031] Ctenopharyngodon idella is a result of 5s rDNA molecular marker. DETAILED DESCRIPTION

[0032] In order to facilitate the understanding of the present application, the following will combine the drawings of the specification and the preferred embodiments to make a more comprehensive and detailed description of the present application, but the protection scope of the present application is not limited to the following specific embodiments.

[0033] Unless otherwise defined, all the professional terms used in the following have the same meaning as that generally understood by those skilled in the art. The professional terms used in the present application are only for the purpose of describing the specific embodiments, and are not intended to limit the protection scope of the present application.

[0034] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0035] Grass carp (Ctenopharyngodon idellus) Cyprinus carpio var. specularis ), Ctenopharyngodon idellus, mirror carp (Cyprinus carpio) Figure 1 Figure 2), Cyprinus carpio Linnaeus. Long-term distant hybridization experiments show that in most cases, the hybrid combination with more chromosomes in the female than in the male cannot obtain surviving fry. Experiments show that grass carp hybrid fry is often significantly different from the appearance of grass carp embryos during embryonic development, such as different degrees of embryo malformation, and the embryo yolk sac always remains spherical, etc., and cannot develop to the feeding period. At the same time, normal fry cannot be obtained without chromosome doubling treatment by using ultraviolet inactivated mirror carp sperm to activate the egg. The reasons for selecting mirror carp as heterologous sperm are as follows: 1. Mirror carp grows faster than ordinary carp under the same breeding conditions; 2. The survival rate of mirror carp is about 98%, the overwintering maturity rate is 96%, and the resistance is strong; 3. The meat quality of mirror carp is better than that of ordinary carp.

[0036] Studies have shown that in the experiment of inducing gynogenesis using heterologous sperm, a small amount of paternal genetic material is integrated into the gynogenetic offspring, and the gynogenetic offspring shows stronger resistance and faster growth rate compared with the ordinary breeding offspring.

[0037] Compared with conventional breeding of carp varieties, mirror carp has the advantages of intact body shape, fast growth rate, high survival rate, low temperature tolerance and strong disease resistance. At the same time, the amount of sperm is sufficient and easy to obtain, and the sperm activity is strong and not easy to be completely inactivated, so the suitable ultraviolet irradiation time (inactivation time is generally 43-52 min, and the specific time is determined by mirror examination results) can be better grasped, and the batch production of gynogenetic grass carp can be better.

[0038] The present application also uses improved red crucian carp sperm and koi carp sperm as heterologous sperm to induce gynogenesis of grass carp and obtains two other gynogenetic grass carps, and sets ordinary grass carp as a control group under the same feeding conditions.

[0039] The feeding time from hatching to the first measurement is 180 days, the feeding time for the second measurement is 360 days, and the feeding time for the third measurement is 540 days. It is found that the gynogenetic grass carp induced by mirror carp sperm is better than the gynogenetic grass carp induced by improved red crucian carp sperm in growth rate and disease resistance, and is better than the gynogenetic grass carp induced by koi carp sperm in growth rate.

[0040] Embodiment:

[0041] The present application uses mirror carp (2n=100) as heterologous sperm to conduct artificial gynogenesis of grass carp in early May, and explores a method for creating fast-growing gynogenetic grass carp, such as Figure 3 , comprising the following steps:

[0042] (1) Mirror carp sperm collection and inactivation: Under the same feeding conditions, two screenings were carried out in half a year as the feeding period. The mirror carp with high specific growth rate (SGR) and good health was selected as the sperm donor, and the SGR formula was used to judge the growth rate. , wherein WB is the weight at the start time (g), WE is the weight at the end time (g), and t is the time length (days). The sperm of the mirror carp was collected by artificial pressing of the abdomen in a light-proof disposable plastic cup, and the sperm motility was detected by a microscope. A small amount of sperm was collected on a glass slide with a toothpick, a small amount of pure water was added to activate the sperm, and the sperm activity time was recorded. The sperm with long survival time and good motility was selected, diluted with 4 times the volume of Hank's solution, mixed well, and stored in a 4 ℃ refrigerator as a "mother liquor". The diluted sperm was spread on the culture dish according to the standard of 0.1-0.2 mm thick, and placed on the ice plate with a rotation speed of 90-100 r / min (a thin towel was placed on the ice plate to increase the friction). Two ultraviolet lamps (model: snowlight ZW20S15W-Z589) with a power of 15 watts were placed 32-35 cm above the culture dish, and the whole device was covered with a black curtain to avoid light. After irradiation for 48 min, the inactivated sperm was collected in a light-proof plastic tube, labeled and stored at 4 ℃. The formula of Hank's solution: NaCl 8-8.2 parts, KCl 0.4-0.45 parts, CaCl2 0.14-0.15 parts, MgSO4·7H2O 0.2-0.25 parts, Na2HPO4·H2O 0.06-0.065 parts, KH2PO4 0.06-0.065 parts, NaHCO3 0.35-0.36 parts, C6H 12 O61.0-1.5 parts, MgCl2·6H2O 0.1-0.15 parts, and water 1000 parts.

[0043] (2) Egg activation: The middle section of the mature grass carp eggs was collected and mixed with the inactivated sperm in step (1), and then stirred with a feather. Then 26 ℃ warm water was added and stirred for 2 min. The fertilized eggs were immediately transferred to a 4 ℃ ice water mixture and continuously stirred for 10-12 min.

[0044] (3) Fertilized egg hatching: The fertilized eggs treated in step (2) for 10-12 min were transferred to 26 ℃ water in batches and stirred gently for 5 min. Then, when the water temperature was basically the same as the normal temperature of the hatching barrel, all the eggs were poured into the hatching barrel.

[0045] (4) Preparation of the fry pond: the fry pond is prepared in advance, the fry pond is disinfected by using quicklime 15 days before the fry is put in, the standard lake water is added and aerated 10 days before the fry is put in, the fry pond is doped by using tea bran, etc. and continuously aerated 8 days before the fry is put in, peanut bran or soybean milk, etc. are added along the edge of the fry pond every morning to carry out the fertilization treatment 2 days before the fry is put in.

[0046] (5) Feeding of the fry: the water flower fry is put into the pre-prepared fry pond to carry out the early feeding, and is transferred into the earth pond to be fed after 3-4 months.

[0047] The present application utilizes the sperm of red crucian carp and mirror carp, and obtains two other kinds of gynogenetic grass carps by using the same method, and carries out the breeding experiment under the same density and feeding conditions, like Figure 4 、 Figure 4 The breeding data of different time periods are shown in Table 1.

[0048] The present application uses three kinds of inactivated sperm to induce the gynogenesis of grass carp eggs, and successfully obtains three kinds of gynogenetic grass carps, and the appearance diagrams are shown in Figure 4 , Figure 4 -A is common grass carp, Figure 4 -B is the gynogenetic grass carp induced by the sperm of red crucian carp, Figure 4 -C is the gynogenetic grass carp induced by the sperm of mirror carp, Figure 5 -D is the gynogenetic grass carp induced by the sperm of mirror carp.

[0049] The present application carries out the preparation observation of the kidney cell chromosomes of two kinds of gynogenetic grass carps and common grass carps, and the steps of the kidney cell chromosome preparation method are as follows: the experimental fish is injected with phytohemagglutinin (PHA) at a dose of 1 mg / mL for 2 times, the interval time of injection is 12-24 h, and colchicine is injected to each sample at a dose of 4-6 ug / g before dissection for 2-6 h. The kidney tissue is ground with 0.8% NaCl, is subjected to hypotonic treatment with 0.075 M KCl at 37°C for 40-60 min, is fixed in 3:1 methanol-acetic acid for 3 times, is dyed with Giemsa dye for 1 hour after dropping, and the metaphase of the chromosomes is observed and photographed by using Pixera Pro 600ES (US), and the results are shown in Figure 5 . The results show that the number of kidney cell chromosomes of common grass carp is 48 ( Figure 5 -A), the number of kidney cell chromosomes of the gynogenetic grass carp induced by the sperm of mirror carp is 48 ( Figure 5 -B), the number of kidney cell chromosomes of the gynogenetic grass carp induced by the sperm of red crucian carp is 48 ( Figure 6 -C), and the number of kidney cell chromosomes of the gynogenetic grass carp induced by the sperm of mirror carp is 48 ( Figure 7 -D). The three kinds of gynogenetic grass carps are all diploid.

[0050] The application uses grass carp male gender-specific molecular markers to identify the gender of common grass carp (1-6), gynogenetic offspring (7-12), and female parent of gynogenetic grass carp (13-18). The tail fins of common grass carp, gynogenetic grass carp, and female parent of gynogenetic grass carp are cut respectively, and the genomic DNA is extracted by using a kit. The PCR amplification is carried out by using the grass carp male-specific gender marker primer, the reaction condition is 94 ℃ pre-denaturation for 5 min, 94 ℃ denaturation for 30 s, 57 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 72 ℃ final extension for 10 min, and 35 cycles are experienced. The PCR product is separated by 1.5% agarose gel electrophoresis, and the electrophoresis results are compared as shown in ​ It is found that sample 3 and sample 6 in common grass carp clone the grass carp male gender-specific molecular marker sequence, and the result is consistent with the observation result of gonad section; the gynogenetic offspring and the female parent of gynogenetic grass carp do not clone the grass carp male gender-specific molecular marker sequence. The results show that the grass carp obtained in the experiment is the gynogenetic offspring.

[0051] The application uses 5s rDNA molecular specific markers to identify the germplasm of gynogenetic grass carp. It is found that the 5s rDNA electrophoretic bands of gynogenetic grass carp are consistent with those of the parent common grass carp.

[0052] The 5s rDNA molecular specific marker method is as follows: the tail fins of common grass carp (1-6), gynogenetic offspring (7-12), and female parent of gynogenetic grass carp (13-18) are cut respectively, and the genomic DNA is extracted by using a kit. The PCR amplification reaction is carried out by using 5s rDNA specific primers (5s-F: 5'-CAGGTTGGTATGGCCGTAAGC-3' and 5S-R: 5'-GCTATGCCCGATCTCGTCTGA-3'). The reaction condition is 94 ℃ pre-denaturation for 5 min, 94 ℃ denaturation for 30 s, 59.2 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 72 ℃ final extension for 10 min, and 35 cycles are experienced. The PCR product is separated by 1.5% agarose gel electrophoresis, and after purification, it is linked to a Pmd18-T vector, and a single clone colony is picked for Sanger sequencing. The electrophoresis results are compared as shown in ​ The 5s rDNA molecular specific marker results of common grass carp, gynogenetic grass carp, and female parent of gynogenetic grass carp are consistent.

[0053] The application uses common grass carp as a control, carries out two kinds of gynogenesis grass carp breeding experiments, under the condition of keeping the pond size, water source, feed, light and the like consistent, the average weight of three kinds of fish is counted every half year (a total of three times), and the survival number after overwintering is counted, as shown in Table 1. The statistical data show that the gynogenesis grass carp induced by mirror carp sperm is superior to the gynogenesis grass carp induced by improved red crucian carp sperm in growth speed and disease resistance, and is superior to the gynogenesis grass carp induced by koi carp sperm in growth speed. Meanwhile, the countable traits of the three kinds of grass carps are counted, and the results are shown in Table 2, and the countable traits are shown in Table 3.

[0054] Table 1 Comparative breeding test data statistics of three kinds of grass carps

[0055]

[0056] Different lower case letters a, b, c in the same column represent significant differences between different fish species P <0.05).

[0057] Table 2 Countable traits of two kinds of gynogenesis grass carps and common grass carps

[0058]

[0059] Table 3 Countable traits of two kinds of gynogenesis grass carps and common grass carps

[0060]

[0061] In summary, the gynogenesis grass carp induced by mirror carp sperm is superior to the gynogenesis grass carp induced by improved red crucian carp sperm and common grass carp in growth speed and disease resistance, and is superior to the gynogenesis grass carp induced by koi carp sperm in growth speed. The application provides a test basis for selection of artificially induced gynogenesis heterologous sperm, and expands the germplasm resources of high-quality grass carps.

Claims

1. A method for creating fast-growing gynogenetic grass carp, characterized in that, It comprises the following steps: (1) screening the sexually mature male mirror carp and the sexually mature female grass carp with obvious growth advantage and stress resistance; (2) collecting the mirror carp sperm and diluting it with Hank's solution, and inactivating the sperm by ultraviolet irradiation; (3) mixing the obtained inactivated sperm with the middle section of the grass carp eggs, and then doubling the treatment by cold shock, and then adding normal temperature water in batches for hatching, and obtaining the gynogenetic grass carp.

2. The method of creating of claim 1, wherein, In step (1), the sexually mature healthy male mirror carp individuals with obvious growth advantage and stress resistance are screened in the mirror carp population by measuring the weight change of the mirror carp with the same age, taking the growth rate as the direct standard for judging the growth advantage of the mirror carp; the sexually mature healthy female grass carp individuals with obvious growth advantage are screened in the grass carp population by measuring the weight change of the grass carp with the same age, taking the growth rate as the direct standard for judging the growth advantage of the grass carp.

3. The method of creating of claim 2, wherein, The growth rate is determined using the specific growth rate (SGR) formula, which is wherein WB is the weight at the start time (g), WE is the weight at the end time (g), and t is the length of the period (days).

4. The method of creating of claim 1, wherein, In step (2), the microscope is used to detect the sperm activity of the mirror carp, and the mirror carp sperm with long survival time and good activity is selected, and the diluted sperm is mixed with 4-5 times the volume of Hank's solution, and the diluted sperm is spread on the culture dish according to the standard of 0.1-0.2 mm thick, and the culture dish containing the sperm is placed on the shaking table with ice bag, and the sperm is inactivated by 2 pieces of 15-watt ultraviolet lamp in the dark environment, the vertical distance between the sperm and the ultraviolet lamp is 30-35 cm, and the irradiation time is 43-52 min.

5. The method of creating according to claim 4, wherein, In step (2), the Hank's solution comprises the following components in parts by mass: NaCl 8-8.2 parts, KCl 0.4-0.45 parts, CaCl20.14-0.15 parts, MgSO4-7H2O 0.2-0.25 parts, Na2HPO4-H2O 0.06-0.065 parts, KH2PO40.06-0.065 parts, NaHCO30.35-0.36 parts, C6H 12 O61.0-1.5 parts, MgCl2-6H2O 0.1-0.15 parts, and water 1000 parts.

6. The method of creating of claim 4, wherein, The rotation speed of the shaking table is 80-120 r / min, and the sperm is kept at 4-6 ℃.

7. The method of creating of claim 4, wherein, The standard for inactivating the sperm is that the microscope detects that the sperm has normal activity of 20-30% when activated by water.

8. The method of creating of claim 1, wherein, In step (3), the obtained inactivated sperm is mixed with the middle section of the grass carp eggs, normal temperature water is added and stirred with a feather, and the fertilized eggs after cold treatment are transferred into 24-26 ℃ warm water for 4-6 min to obtain the doubled treated fertilized eggs.

9. The method of creating of claim 8, wherein, The doubled treated fertilized eggs are added into normal temperature water in batches, and when the water temperature is consistent with the water temperature in the hatching device, they are transferred into the hatching device for normal hatching, and the obtained fry after hatching is transferred to the pre-fertilized cement pool for early feeding, and the gynogenetic grass carp is obtained.

10. The method of engineering of any one of claims 1-9, wherein, The hatching is flow hatching, and the water flow is suitable for rolling the fertilized eggs, and the water temperature of the hatching barrel is 22-26 ℃.

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