Breeding method of new ricefield eel strain with high disease resistance

Abstract

The invention discloses a breeding method of a new ricefield eel strain with high disease resistance. The method comprises the following steps: step 1, selecting a breeding group; step 2, screening of breeding parents; step 3, establishment of a breeding base population; 4, screening a parent F0 generation of the breeding base population; 5, establishing and screening an F1 generation; 6, reverse selection of the generation F0; 7, establishing and screening an F2 generation; 8, reverse selection of the F1 generation; 9, establishing and screening an F3 generation; and 10, carrying out group verification. According to the breeding method for the new ricefield eel strain with the high disease resistance, the immunity and the disease resistance of the ricefield eel fries can be enhanced, the survival rate of the ricefield eel fries is increased, the technical regulation of ricefield eel fry breeding is formulated, and healthy development of the ricefield eel breeding industry is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic breeding, and specifically relates to a method for breeding a new strain of yellow eel with strong disease resistance. Background Art

[0003] At present, the domestic eel market is dominated by wild eels. Although there have been some technological breakthroughs in artificial breeding in recent years, the proportion is still small. Since the beginning of eel breeding, the most prominent problem is the high mortality rate caused by low immunity and poor disease resistance of seedlings. It is urgent to solve this industry problem through variety breeding. Summary of the invention

[0004] The present invention provides a method for breeding a new strain of yellow eel with strong disease resistance, which specifically comprises the following steps:

[0005] Step 1: Selection of breeding population: RAD-seq sequencing analysis was performed on 137 wild eel samples of different sexes and growth stages collected from 19 populations across the country; Chongming (CM), Weishan Lake (WSH), Huoqiu (HQ), Jiaoling (JL) and Lingshui (LS) populations were selected from the 19 populations of wild eels according to the genetic evolutionary relationship of eels across the country, and the Chongming population was used as the local control population;

[0006] Step 2: Screening of breeding parents: The eels from Chongming, Gushan, Huoqiu, Jiaoling and Lingshui were screened four times respectively, and those that did not meet the standards were eliminated;

[0007] Step 3: Establishment of the basic breeding population: Select female eels (15.7-18.3g) and male eels (38.1-42.9g) of similar size from the above five populations, and take liver and serum in the laboratory for antioxidant and immune enzyme activity detection; At the same time, artificial injection of Aeromonas hydrophila was performed on the eels of the five populations, and the eels of the five populations were placed in 90L blue polyethylene boxes for continued breeding and bleeding was observed; According to the summary and analysis of the experimental results, the Huoqiu (HQ) population with the strongest disease resistance was selected as the basic breeding population;

[0008] Step 4: Screening of the F0 generation of the breeding base population: Randomly select healthy, disease-free, and well-developed female eels (47.2±1.7g) and male eels (75.8±2.1g) from the Huoqiu (HQ) population as the F0 generation of parents;

[0009] Step 5: Establishment and screening of the F1 generation: Through the precise pairing technology of yellow eels, the F0 generation is paired with males and females in a 1:1 ratio to obtain the F1 generation, and the top 200 female eels of the F1 generation in terms of growth rate are selected as the parents of the F2 generation;

[0010] Step 6: Back-selection of F0 generation: Since the yellow eel has a special physiological phenomenon of sex reversal, the female eels of the F0 generation will be reversed to male eels after breeding. Therefore, the male eel parents of the F2 generation will be backcrossed with the selected F1 generation female eels;

[0011] Step 7: Establishment and screening of the F2 generation: Through the precise pairing technology of yellow eels, the female eels of the F1 generation and the male eels of the F0 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F2 generation. The top 200 female eels of the F2 generation in terms of growth rate are selected as the parents of the F3 generation;

[0012] Step 8: Counter-selection of F1 generation: Reverse the reproductive sex of F1 generation to male eels as the parents of F3 generation, and backcross them with the selected F2 generation female eels;

[0013] Step 9: Establishment and screening of the F3 generation: Through precise pairing technology of yellow eels, female eels of the F2 generation and male eels of the F1 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F3 generation;

[0014] Step 10: Cultivation and propagation: Carry out large-scale cultivation and propagation of F3 generation seedlings.

[0015] The disease resistance mentioned herein refers to the activities of superoxide dismutase (SOD), acid phosphatase (ACP), malondialdehyde (MDA), phenoloxidase (PO); and the bleeding condition after attacking Aeromonas hydrophila.

[0016] Furthermore, the four screening methods in step 2 are: appearance screening, behavior screening one, behavior screening two, and food screening.

[0017] Further, the four screening methods are specifically implemented as follows:

[0018] First screening (appearance screening): separate the eels by size, check whether they are sick (eliminate sick eels), and separate the rest by size and put them into plastic boxes or screening pools;

[0019] Second screening (behavioral screening 1): Observe in plastic boxes or screening pools from 12 noon to 2 pm. The eels that climb up the fish tank, raise their heads, or move around alone are poor quality fish and should be removed and eliminated.

[0020] The third screening (behavioral screening 2): At about 6 pm on the same day, the disinfected eels were placed in the eel pond near the feeding table. At noon the next day, those that came out to move alone, went to the grass, or raised their heads were selected and eliminated;

[0021] The fourth screening (food screening): if the eels eat more than 1% of their body weight (calculated according to their body weight) on at least one day for 5 consecutive days, they will be kept; if the eels eat less than 1% of their body weight for 5 consecutive days, they will be eliminated.

[0022] The present invention provides a method for breeding a new strain of yellow eel with strong disease resistance, which has the following beneficial effects:

[0023] 1. The new strain of eel bred in this scheme can effectively enhance the immunity and disease resistance of its seedlings and improve the survival rate compared with the parent strain;

[0024] 2. The breeding method provided in this plan has formulated technical regulations for the cultivation of eel seedlings to ensure the healthy development of the eel breeding industry.

[0025] In view of the shortcomings of the existing technology, the present invention provides a method for breeding a new strain of eel with strong disease resistance, which solves the problem of high mortality rate caused by low immunity and poor disease resistance of eel seedlings, and ensures the healthy development of the eel breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention provides a schematic flow chart of a method for breeding a new strain of rice field eels with strong disease resistance.

[0027] Figure 2 SOD activities in serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) of five breeding groups of yellow field eels.

[0028] Figure 3 ACP activities in serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) of five breeding populations of yellow field eel.

[0029] Figure 4 MDA activities in serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) of five breeding populations of yellow field eel.

[0030] Figure 5 PO activities in serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) of five breeding groups of yellow field eels.

[0031] Figure 6 Bleeding in five selected populations of yellowtail eels after injection with Aeromonas hydrophila. DETAILED DESCRIPTION

[0032] The following examples are only used to further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods without specifying specific conditions in the examples and the reagents and materials without specifying the formula are all in accordance with the conventional conditions in the art, and the reagents used can all be purchased.

[0033] Embodiment 1 The breeding method of the new strain of rice field eel with strong disease resistance specifically comprises the following steps:

[0034] Step 1: Selection of breeding population: RAD-seq sequencing analysis was performed on 137 wild eel samples of different sexes and growth stages collected from 19 populations across the country (sequencing was performed by Shanghai Shenggong Biotechnology Co., Ltd.); Based on the genetic evolutionary relationship of eels across the country, Chongming (CM), Weishan Lake (WSH), Huoqiu (HQ), Jiaoling (JL) and Lingshui (LS) populations were selected from the 19 populations of wild eels for follow-up research, and the Chongming population was used as the local control population; (References for the method of selecting breeding populations: Asian Swamp eel Monopterus albus Population Structure and Genetic Diversity in China, Weiwei Lv et al., Frontiers in Genetics.)

[0035] Step 2: Screening of breeding parents: The eels from Chongming, Gushan, Huoqiu, Jiaoling and Lingshui were screened four times respectively, and those that did not meet the standards were eliminated;

[0036] The four systematic screening methods are: appearance screening, behavior screening 1, behavior screening 2, and food screening;

[0037] First screening (appearance screening): separate the eels by size, check whether they are sick (eliminate sick eels), and put them into plastic boxes or screening pools after separation;

[0038] Second screening (behavioral screening 1): Observe in plastic boxes or screening pools from 12 noon to 2 pm. If the eels climb up the fish tank, raise their heads, or move around alone, they are poor quality fish and should be removed and eliminated.

[0039] The third screening (behavioral screening 2): At about 6 pm on the same day, the disinfected eels were placed in the eel pond near the feeding table. At noon the next day, those that came out to move alone, went to the grass, or raised their heads were selected and eliminated;

[0040] The fourth screening (food screening): if the eels eat more than 1% (weight percentage) of their body weight on at least one day out of 5 consecutive days, the eels will be kept; if the eels eat less than 1% of their body weight for 5 consecutive days, they will be eliminated.

[0041] Step 3: Establishment of the basic breeding population: 10 female eels (15.7-18.3 g) and 10 male eels (38.1-42.9 g) of similar size were selected from the above five populations, and the liver and serum were taken in the laboratory for antioxidant and immune enzyme activity detection; at the same time, 50 yellow eels (25 females and 25 males) were taken from each of the five populations, and artificially injected with Aeromonas hydrophila. The yellow eels of the five populations were placed in 90L blue polyethylene boxes for continued breeding and bleeding was observed; based on the summary and analysis of the experimental results, the Huoqiu (HQ) population was finally selected as the basic breeding population; the specific experimental steps and experimental results are as follows:

[0042] (1) Detection of superoxide dismutase (SOD) activity in five breeding populations.

[0043] Ten female eels (15.7-18.3 g) and ten male eels (38.1-42.9 g) of similar size were randomly selected from five breeding populations of wild eels in Chongming (CM), Weishanhu (WSH), Huoqiu (HQ), Jiaoling (JL) and Lingshui (LS). Partial liver tissue and serum were obtained by dissection. The tissue was fully ground and broken and diluted with physiological saline to obtain a 10% tissue homogenate. The liver and serum were tested using a SOD enzyme activity detection kit (Cat. No.: A001-3, Brand: Nanjing Jiancheng).

[0044] The experimental results are as follows Figure 2 As shown, the serum SOD activity of male eels in WSH and HQ groups was significantly higher than that in other groups (P<0.05). The serum SOD activity of female eels in WSH group was significantly higher than that in CM and LS groups (P<0.05), while there was no significant difference in the SOD activity of female eels in HQ and JL groups with the other three groups (P>0.05). The liver SOD activity of male eels in HQ group was significantly higher than that in CM and LS groups (P<0.05), while there was no significant difference in the SOD activity of male eels in WSH and JL groups with the other three groups (P>0.05). The liver SOD activity of female eels in HQ group was significantly higher than that in the other four groups (P<0.05). The serum SOD activities of male and female eels in the WSH group were 65.9% and 114.3% higher than those in the local group (CM), respectively; the serum SOD activities of male and female eels in the HQ group were 85.9% and 64.8% higher than those in the local group (CM), respectively; the liver SOD activities of male and female eels in the WSH group were 71.6% and 21.6% higher than those in the local group (CM), respectively; the liver SOD activities of male and female eels in the HQ group were 32.8% and 104.8% higher than those in the local group (CM), respectively.

[0045] (2) Detection of acid phosphatase (ACP) activity in five breeding populations.

[0046] Ten female eels (15.7-18.3 g) and ten male eels (38.1-42.9 g) of similar size were randomly selected from five breeding populations of wild eels in Chongming (CM), Weishanhu (WSH), Huoqiu (HQ), Jiaoling (JL) and Lingshui (LS). Partial liver tissue and serum were obtained by dissection. The tissue was fully ground and broken and diluted with physiological saline to obtain a 10% tissue homogenate. The liver and serum were tested using the ACP enzyme activity detection kit (Cat. No.: A060-2, Brand: Nanjing Jiancheng).

[0047] The experimental results are as follows Figure 3 As shown, the serum ACP activity of male and female eels in WSH and HQ groups was significantly higher than that in the other three groups, while the serum ACP activity of female eels in CM was significantly higher than that in JL and LS groups (P<0.05). The liver ACP activity of female and male eels in HQ group was significantly higher than that in the other four groups (P<0.05), while the liver ACP activity of female eels in CM and WSH groups was significantly higher than that in JL and LS groups (P<0.05). The serum ACP activity of male and female eels in HQ group was 63.1% and 98.3% higher than that in the local group (CM), respectively; the serum ACP activity of male and female eels in WSH group was 93.0% and 190.7% higher than that in the local group (CM), respectively; the liver ACP activity of male and female eels in HQ group was 168.4% and 76.7% higher than that in the local group (CM), respectively.

[0048] (3) Detection of malondialdehyde (MDA) activity in five breeding populations.

[0049] Ten female eels (15.7-18.3 g) and ten male eels (38.1-42.9 g) of similar size were randomly selected from five breeding populations of wild eels in Chongming (CM), Weishanhu (WSH), Huoqiu (HQ), Jiaoling (JL) and Lingshui (LS). Partial liver tissue and serum were obtained by dissection. The tissue was fully ground and broken and diluted with physiological saline to obtain 10% tissue homogenate. The liver and serum were tested using the MDA activity detection kit (Cat. No.: A003-1, Brand: Nanjing Jiancheng).

[0050] The experimental results are as follows Figure 4As shown, the serum MDA activity of male and female eels in the HQ group was significantly higher than that in the WSH, JL and LS groups (P<0.05), while the serum MDA activity of male eels in the CM group had no significant difference with the other four groups (P>0.05). The liver MDA activity of male and female eels in the WSH and HQ groups was significantly higher than that in the CM, JL and LS groups (P<0.05). The serum MDA activity of male and female eels in the HQ group was 50.0% and 97.5% higher than that in the local group (CM), respectively; the liver MDA activity of male and female eels in the WSH group was 93.1% and 98.6% higher than that in the local group (CM), respectively; the liver MDA activity of male and female eels in the HQ group was 159.2% and 76.6% higher than that in the local group (CM), respectively.

[0051] (4) Detection of phenoloxidase (PO) activity in five breeding populations.

[0052] Ten female eels (15.7-18.3 g) and ten male eels (38.1-42.9 g) of similar size were randomly selected from five breeding populations of wild eels in Chongming (CM), Weishanhu (WSH), Huoqiu (HQ), Jiaoling (JL) and Lingshui (LS). Partial liver tissue and serum were obtained by dissection. The tissue was fully ground and broken and diluted with physiological saline to obtain a 10% tissue homogenate. The liver and serum were tested using the PO activity detection kit (Cat. No.: H247-1, Brand: Nanjing Jiancheng).

[0053] The experimental results are as follows Figure 5 As shown in the results, there was no significant difference in serum and liver PO activity of female and male eels in the five groups (P>0.05). In terms of serum PO activity, the serum PO activity of male eels in the WSH group and female eels in the LS group was slightly higher than that of the other groups. In terms of liver PO activity, the liver PO activity of female and male eels in the JL group was significantly higher than that of the other groups. The serum PO activity of female eels in the WSH and HQ groups was 60.0% higher than that of the local group (CM); the liver PO activity of male eels in the WSH and HQ groups was 33.2% and 42.4% higher than that of the local group (CM), respectively.

[0054] (5) Detection of bleeding in five selected populations challenged with Aeromonas hydrophila.

[0055] 25 female eels and 25 male eels of similar size were randomly selected from the five breeding populations of Chongming (CM), Weishan Lake (WSH), Huoqiu (HQ), Jiaoling (JL) and Lingshui (LS) and artificially injected with Aeromonas hydrophila (purchased from Beina Biotechnology). Before injection, the strain was cultured in LB medium at 28°C and 150 rpm for 24 hours, centrifuged at 6000 rpm in a low-temperature centrifuge for 5 minutes, then washed with PBS and centrifuged again, and repeated twice. After plate counting, the concentration of the bacterial suspension was adjusted to 1×10 6 cfu / mL, and after injecting 200 μL of Aeromonas hydrophila suspension into each eel, the eel was placed in a 90L blue polyethylene box for continued culture and the bleeding was observed.

[0056] The experimental results are as follows Figure 6 As shown in the figure, all eels in the CM, LS and JL groups bled and eventually died 3, 4 and 5 days after the Aeromonas hydrophila attack, respectively. In the HQ group, only 18 eels bled and died 5 days after the Aeromonas hydrophila attack, and 15 eels survived 7 days later. In the WSH and HQ groups, the number of bleeding eels was 41 and 35, respectively, 7 days after the Aeromonas hydrophila attack.

[0057] From the five experiments, it can be seen that the antioxidant and immune enzyme activities of female and male eels in the Huoqiu (HQ) population are generally higher. Moreover, the vast majority of enzyme activities in the Huoqiu (HQ) population are 5% higher than those in the local Chongming (CM) population. At the same time, after injection of Aeromonas hydrophila, all eels in the Chongming (CM), Jiaoling (JL) and Lingshui (LS) populations showed bleeding within 5 days, while some eels in the Weishan Lake (WSH) and Huoqiu (HQ) populations did not bleed 7 days after the infection. Therefore, it can be preliminarily concluded that the eels in the Weishan Lake (WSH) and Huoqiu (HQ) populations have a strong ability to resist hemorrhagic disease, and the Huoqiu (HQ) population with the best performance was selected as the basic population for breeding.

[0058] The experimental data were expressed as mean ± standard error (mean ± SE). The data were calculated using Microsoft Excel and analyzed using SPSS22.0 software. The significant differences between the groups were evaluated by one-way analysis of variance and Duncan's multiple comparisons, and P values ​​< 0.05 were considered statistically significant.

[0059] Step 4: Screening of the F0 generation of the breeding base population: Randomly select 150 female eels (47.2±1.7g) and male eels (75.8±2.1g) with healthy physique, no disease or injury, and good gonadal development from the Huoqiu (HQ) population as the F0 generation of parents;

[0060] Step 5: Establishment and screening of the F1 generation: Through the precise pairing technology of yellow eels, the F0 generation is paired with males and females in a 1:1 ratio to obtain the F1 generation, and the top 200 female eels of the F1 generation in terms of growth rate are selected as the parents of the F2 generation;

[0061] Step 6: Back-selection of F0 generation: Since the yellow eel has a special physiological phenomenon of sex reversal, the female eels of the F0 generation will be reversed to male eels after breeding. Therefore, the male eel parents of the F2 generation will be backcrossed with the selected F1 generation female eels;

[0062] Step 7: Establishment and screening of the F2 generation: Through the precise pairing technology of yellow eels, the female eels of the F1 generation and the male eels of the F0 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F2 generation. The top 200 female eels of the F2 generation in terms of growth rate are selected as the parents of the F3 generation;

[0063] Step 8: Counter-selection of F1 generation: Reverse the reproductive sex of F1 generation to male eels as the parents of F3 generation, and backcross them with the selected F2 generation female eels;

[0064] Step 9: Establishment and screening of the F3 generation: Through precise pairing technology of rice field eels, female eels of the F2 generation and male eels of the F1 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F3 generation, that is, a new rice field eel strain with strong disease resistance;

[0065] Step 10: Cultivation and propagation: Carry out large-scale cultivation and propagation of F3 generation seedlings.

[0066] Example 2 Verification of disease resistance of the new strain of eel bred in Example 1

[0067] The experimental method refers to the steps in step 3 of Example 1, and the experimental results are shown in Table 1:

[0068] Table 1 Results of detection of several enzyme activities in serum and liver of new strain of rice field eel

[0069]

[0070]

[0071] Seven days after the infection with Aeromonas hydrophila, the number of hemorrhagic eels was 24, which was a 31.4% lower incidence rate than that of the parent.

[0072] Three other groups of new strains of rice field eels were randomly selected for testing of SOD, ACP, MDA and PO activities, and the results were stable, with a difference of 10% from the data in Table 1. This shows that the new strain of rice field eels selected and harvested in Example 1 has stable disease resistance after large-scale cultivation and expansion.

Claims

1. A method for breeding a new strain of rice field eel with strong disease resistance, characterized in that The steps include: Step 1: Selection of breeding population: RAD-seq sequencing analysis was performed on 137 wild eel samples of different sexes and growth stages collected from 19 populations across the country; Chongming CM, Weishan Lake WSH, Huoqiu HQ, Jiaoling JL and Lingshui LS populations were selected from the 19 populations of wild eels, and the Chongming population was used as the local control population; Step 2: Screening of breeding parents: The eels from Chongming, Gushan, Huoqiu, Jiaoling and Lingshui were screened four times respectively, and those that did not meet the standards were eliminated; Step 3: Establishment of the basic breeding population: Select female eels 15.7-18.3g and male eels 38.1-42.9g of similar size from the above five populations, and take liver and serum in the laboratory for antioxidant and immune enzyme activity detection; At the same time, artificially inject Aeromonas hydrophila into the eels of the five populations, place the eels in blue polyethylene boxes for continued breeding and observe bleeding; According to the summary and analysis of the experimental results, select the Huoqiu HQ population with the strongest disease resistance as the basic breeding population; Step 4: Screening of the F0 generation of the breeding base population: Randomly select healthy, disease-free, and well-developed female eels weighing 47.2±1.7g and male eels weighing 75.8±2.1g from the Huoqiu HQ population as the F0 generation of parents; Step 5: Establishment and screening of the F1 generation: Through the precise pairing technology of yellow eels, the F0 generation is paired with males and females in a 1:1 ratio to obtain the F1 generation, and the top 200 female eels of the F1 generation in terms of growth rate are selected as the parents of the F2 generation; Step 6: Back-selection of F0 generation: Since the yellow eel has a special physiological phenomenon of sex reversal, the female eels of the F0 generation will be reversed to male eels after breeding. Therefore, the male eel parents of the F2 generation will be backcrossed with the selected F1 generation female eels; Step 7: Establishment and screening of the F2 generation: Through the precise pairing technology of yellow eels, the female eels of the F1 generation and the male eels of the F0 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F2 generation. The top 200 female eels of the F2 generation in terms of growth rate are selected as the parents of the F3 generation; Step 8: Counter-selection of F1 generation: Reverse the reproductive sex of F1 generation to male eels as the parents of F3 generation, and backcross them with the selected F2 generation female eels; Step 9: Establishment and screening of the F3 generation: Through precise pairing technology of yellow eels, female eels of the F2 generation and male eels of the F1 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F3 generation; Step 10: Cultivation and propagation: Carry out large-scale cultivation and propagation of F3 generation seedlings.

2. The method for breeding a new strain of rice field eels with strong disease resistance according to claim 1, wherein the four screening methods in step 2 are: appearance screening, behavior screening 1, behavior screening 2, and food screening: Appearance screening: sort the eels by size and check whether they are sick. Eliminate sick eels and separate the rest by size and put them into plastic boxes or screening pools; Behavioral screening 1: Observe in a plastic box or screening pool from 12 noon to 2 pm. The eels that climb up the fish tank, raise their heads, or move around alone are poor quality fish and should be removed and eliminated. Behavioral screening 2: At around 6 pm on the same day, the disinfected eels were placed into the eel pond near the feeding table. At noon the next day, those that came out to move alone, went to the grass, or raised their heads were selected and eliminated; Food screening: If the eels eat more than 1% of their body weight on at least one day out of five consecutive days, the eels will be kept; Eels that eat less than 1% of their body weight for 5 consecutive days will be eliminated.

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