A method for cultivating a new variety of Pinctada fucata martensii rich in carotenoids

Abstract

The present invention discloses a method for cultivating a new variety of Pinctada fucata martensii rich in carotenoids. Based on measuring the carotenoid content in the adductor muscle, the method uses the family gene pyramiding breeding technique to cultivate a new variety of Pinctada fucata martensii rich in carotenoids, including the following steps: a. Collection of germplasm resources; b. Construction of basic families; c. Selection of parents; d. Construction of core families - second-generation families; e. Construction of core families - third-generation families; f. Continuing to construct core families according to the method in step e for more than 5 generations, and finally obtaining a new variety of Pinctada fucata martensii rich in carotenoids. The new variety of Pinctada fucata martensii cultivated by the present invention has the advantages of strong stress resistance and fast growth rate, effectively solving the problems such as individual miniaturization and high mortality rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of cultivating new varieties of marine shellfish, and particularly relates to a method for cultivating a new variety of Pinctada fucata rich in carotenoids. Background Art

[0002] Pinctada fucata is an important marine cultured shellfish and the main mother shell for producing pearls. The well-known "South Sea Pearls" are produced by this kind of shellfish. Pinctada fucata lives in tropical and subtropical sea areas. The research and production of cultivating pearls from Pinctada fucata in China started in the mid-20th century. The artificial pearl cultivation was successful in 1958, and the artificial seedling breeding was successful in 1965, marking the entry of the cultivation of Pinctada fucata into the era of full artificial cultivation, that is, using the seedlings artificially propagated for cultivation, and then selecting mature individuals for artificial reproduction after cultivation, forming a closed cycle. After more than 40 years of artificial reproduction and cultivation, due to the lack of scientific selective breeding, problems such as individual miniaturization and high mortality rate have emerged, indicating a decline in stress resistance and germplasm degradation.

[0003] During the 10th Five-Year Plan and 11th Five-Year Plan periods, selective breeding research on Pinctada fucata martensii was carried out in China, and four new varieties, namely NanZhen No. 1, ZhongKe No. 1, HaiDa No. 1 and HaiXuan No. 1, were obtained respectively.The applicant's supervisor has also carried out relevant research on the selective breeding of Pinctada fucata martensii and achieved corresponding results. For example, the applicant's supervisor applied for "A Method for Cultivating a New Variety of Small Pinctada fucata martensii" on August 31, 2020, with the patent application number "202010900146.7". This achievement was mainly proposed to address the technical problem in the prior art that "in the production practice of pearl oyster nucleus insertion, the pearl oysters and small Pinctada fucata martensii generally come from the same batch of oysters without specialized breeding, resulting in weak ability to secrete nacre in the mantle small pieces, mixed colors, and low gloss, seriously affecting the quality of pearls and restricting the development of the pearl farming industry". It mainly includes the following steps: (1) Establishment of the base population: Select 1000 oysters with regular body shapes, relatively large sizes, and thick shells from a certain wild Pinctada fucata martensii population at a selection intensity of 10% as candidate parent oysters. (2) Selection of parent oysters: By measuring the phenotypic data of the 1000 candidate parent oysters, calculate their body mass coefficients, and select the top 200 oysters with larger body mass coefficients. Open the shells through dissection and select oysters with plump gonad development, white nacre color on the inner shell of the shell, good gloss, and no defects as parents. (3) Obtaining gametes: After dissecting the parent oysters, pick a small amount of gonad with a toothpick, and select 30 mature female and male parents each through microscopic examination. Put the eggs and sperm of each individual into 500 ml beakers respectively. (4) Constructing the first-generation selected population F1: a. Mating and hatching: First, mix the eggs in the 30 beakers together, then evenly distribute the obtained mixed egg liquid into another 30 beakers. Activate the egg liquid in the beakers respectively, activate the sperm of the 30 dissected male parents and add them to the activated eggs respectively. After fertilization, transfer them to an incubation bucket, filter out debris with a 300-mesh silk screen, and then filter out excess sperm with a 500-mesh silk screen to prevent polyspermy. b. Larval cultivation: Let the above fertilized eggs develop in water for 20 - 24 hours to obtain D-shaped larvae, then mix them equally and culture them in a cultivation pond for 45 - 60 days to obtain 2 mm juvenile oysters. c. Adult oyster cultivation: Put the above-cultured juvenile oysters into the sea for cultivation to obtain F1-generation adult oysters; construct more than 3 populations using the above method. (5) Constructing the selected F2-generation population: Select candidate parents according to the method described in step (1), calculate the body mass coefficient according to the method described in step (2), and estimate the breeding value of the body mass coefficient using the BLUP method. Select the parents with higher breeding values and mate them in the following way: 30 female parents in population A are respectively mated with 30 male parents in population B, 30 female parents in population B are respectively mated with 30 male parents in population C, and 30 female parents in population C are respectively mated with 30 male parents in population A to construct three F2-generation selected populations. The specific cultivation method is as described in step (4). (6) Repeat step (5) and conduct successive generations of selection for more than 4 generations.The cultivation method of the new variety of small piece shells of Pinctada fucata martensii does not require the introduction of foreign genes through complex technologies such as genetic engineering. On the premise of having no impact on biological and environmental safety, through specialized breeding, a new variety of small piece shells of Pinctada fucata martensii with strong nacre secretion ability and high luster of nacre layer is provided, making up for the deficiencies of the existing technology.

[0004] During the continuous research process, the applicant found that Pinctada fucata martensii rich in carotenoids has better stress resistance and growth rate than ordinary Pinctada fucata martensii; at the same time, there is also a blank in the research on new varieties of Pinctada fucata martensii rich in carotenoids. Summary of the Invention

[0005] The purpose of the present invention is to provide a cultivation method for a new variety of Pinctada fucata martensii rich in carotenoids. This method is based on measuring the carotenoid content in the adductor muscle, and uses the family gene pyramiding breeding technology to cultivate a new variety of Pinctada fucata martensii rich in carotenoids. This new variety of Pinctada fucata martensii has the advantages of strong stress resistance and fast growth rate, effectively solving problems such as individual miniaturization and high mortality.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A cultivation method for a new variety of Pinctada fucata martensii rich in carotenoids successively includes the following steps:

[0008] a. Collection of germplasm resources:

[0009] Collect Pinctada fucata martensii and transport them back to the farm for temporary cultivation until the breeding season;

[0010] b. Construction of basic families:

[0011] Dissect the Pinctada fucata martensii collected in step a, measure the carotenoid content of individuals with red, orange-red or light red adductor muscles, and select individuals with a content > 120 μg / g as parents to construct full-sib families according to the male-female ratio of 1:1;

[0012] Each family is cultivated and managed under the same conditions;

[0013] After the seedlings emerge, they are cultured and managed under the same conditions in the same sea area. When they are cultured for one year, trait measurements are carried out. For each family, more than 1000 male and female individuals are measured; the measured trait indicators include shell length, shell height, and carotenoid content;

[0014] Then use the ASReml software to calculate the breeding value and heritability parameters based on the BLUP animal model;

[0015] c. Selection of parents:

[0016] Select the families with breeding values higher than the average according to the ranking of the breeding values of carotenoid content in the above pedigrees as candidate families for seed selection;

[0017] Rank according to the average breeding values of males and females and number them in order. The one with the largest breeding value is numbered 1, and so on;

[0018] Before the breeding season, conduct individual screening of the selected families by gender: For each family, select the top 200 individuals of both genders according to size for trait measurement and mark each individual. Then calculate the breeding value of each individual and rank them. The individuals with rankings higher than the average are used as candidate parents respectively;

[0019] d. Construction of the core family - the second-generation family:

[0020] Select the female individual with the highest individual breeding value from the family with the highest female breeding value and the male individual with the highest individual breeding value from the family with the highest male breeding value for mating in a 1:1 ratio; then select the female individual with the highest individual breeding value from the family with the second-highest female breeding value and the male individual with the highest individual breeding value from the family with the second-highest male breeding value for mating in a 1:1 ratio; and so on, complete the pairing among all the selected families, combining the best with the best, to achieve the purpose of aggregating excellent genes for qualitative traits;

[0021] The methods and steps of reproduction, seedling cultivation and aquaculture are the same as those in step b;

[0022] When the aquaculture reaches one year, conduct trait measurement and calculate the family breeding value and heritability parameters. The trait indicators measured and the calculation methods of the family breeding value and heritability parameters are the same as those in step b;

[0023] e. Construction of the core family - the third-generation family:

[0024] According to the methods in step c and step d, first rank the average breeding values of males and females among families, and then rank the breeding values of males and females within families. On the premise of avoiding inbreeding, select the best female individual from the best female family and the best male individual from the best male family for mating, the best male and female individuals from the families ranked second for mating, and so on, to construct the third-generation core family, and so on, complete the pairing among all families, further aggregating excellent genes; during the mating process, avoid mating between male and female individuals from the same family. If the inbreeding coefficient of the paired individuals reaches the set threshold of 1, they are respectively mated with individuals from the next ranked family;

[0025] f. Continue to construct the core family according to the method in step e until more than 5 generations, and finally obtain a new variety of Pinctada fucata rich in carotenoids.

[0026] The present invention is further illustrated as follows. In step a, more than 2000 parents are collected from at least 3 different sea areas, different farms or different strains, and individuals with regular, complete appearance and no diseases are selected.

[0027] The present invention is further illustrated as follows. In step b, the seedling breeding of all families is carried out under the same conditions. When the seedlings reach about 1 mm, they are transplanted to the sea area for cage culture with the same stocking density.

[0028] Advantages of the present invention:

[0029] 1. Since most economic traits are quantitative traits, but there is no breeding for qualitative traits of specialized strains at present, quantitative traits and qualitative traits are usually controlled by many genes, and the effects of different genes vary. Therefore, the cultivation method of the present invention can aggregate excellent genes on the same individual at the fastest speed, effectively improving the breeding efficiency. In addition, inbreeding will cause inbreeding depression, which is an important factor leading to breeding failure. The cultivation method of the present invention can effectively control the degree of inbreeding, prevent inbreeding depression, and thus breed a new variety of pearl oyster rich in natural antioxidants with excellent traits and stable heredity.

[0030] 2. The new variety of Pinctada fucata martensii cultivated by the cultivation method of the present invention has the advantages of strong stress resistance and fast growth rate, effectively solving the problems of individual miniaturization and high mortality.

[0031] 3. According to the calculation of experimental data, the pearl oysters bred with the new variety of Pinctada fucata martensii selected by the present invention show good pearl-producing traits; moreover, the survival rate of the selected new variety of Pinctada fucata martensii in cultivation is increased by 24.77% compared with that of ordinary Pinctada fucata martensii, the carotenoid content is increased by 125%, and the average shell height and shell length of one-year-old shellfish are increased by 27.27% and 25% respectively.

[0032] Table 1. Comparison between the new variety and ordinary Pinctada fucata martensii.

[0033] Shellfish species Cultivation survival rate Carotenoid content (ug / g) Shell length (mm) Shell height (mm) New variety of Pinctada fucata 80.1% 176.2 75 70 Common Pinctada fucata 64.2% 78.3 60 55 Brief Description of the Drawings

[0034] Figure 1 It is a schematic flow chart of the gene aggregation breeding technology for shellfish families. Detailed Embodiments

[0035] The present invention will be further illustrated below with reference to the drawings.

[0036] Example:

[0037] A method for cultivating a new variety of Pinctada fucata martensii rich in carotenoids successively includes the following steps:

[0038] a. Collection of germplasm resources:

[0039] Collect Pinctada fucata martensii and transport them back to the farm for temporary cultivation until the breeding season;

[0040] Collect more than 2,000 parent individuals of Pinctada fucata martensii from at least 3 different sea areas, different farms or different strains, and select individuals with regular, complete appearance and no diseases;

[0041] b. Construction of basic families:

[0042] Dissect the Pinctada fucata martensii collected in step a, measure the carotenoid content of individuals with red, orange-red or light red adductor muscles, and select individuals with a content > 120 μg / g as parents to construct full-sib families according to the male-female ratio of 1:1;

[0043] Each family is cultivated and managed under the same conditions;

[0044] After the seedlings emerge, they are cultured and managed under the same conditions in the same sea area. At the first anniversary of cultivation, trait measurements are taken, and more than 1,000 male and female individuals are measured for each family; the measured trait indicators include shell length, shell height, and carotenoid content;

[0045] Then use ASReml software to calculate breeding values and heritability parameters based on the BLUP animal model;

[0046] c. Selection of parents:

[0047] Select families with breeding values higher than the average according to the ranking of the above family breeding values as candidate families for seed retention;

[0048] Rank according to the average breeding values of males and females and number them in order. The family with the largest breeding value is numbered 1, and so on;

[0049] Before the breeding season, screen individuals of the selected families by male and female respectively: For each family, select the top 200 male and female individuals according to size for trait measurement and mark each individual, then calculate the breeding value of each individual and rank them. Individuals with a ranking greater than the average are used as candidate parents respectively;

[0050] d. Construction of core families - second-generation families:

[0051] Select the female individual with the first-ranked individual breeding value from the family with the first-ranked female breeding value and the male individual with the first-ranked individual breeding value from the family with the first-ranked male breeding value for mating at a ratio of 1:1; then select the female individual with the first-ranked individual breeding value from the family with the second-ranked female breeding value and the male individual with the first-ranked individual breeding value from the family with the second-ranked male breeding value for mating at a ratio of 1:1; and so on, complete the pairing between all selected families, combine the best with the best, and achieve the purpose of aggregating excellent genes for qualitative traits;

[0052] The methods and steps for reproduction, seedling cultivation, and breeding are the same as those in step b;

[0053] When breeding for one year, trait measurements and calculations of family breeding values and heritability parameters are carried out. The measured trait indicators and the calculation methods for family breeding values and heritability parameters are the same as those in step b;

[0054] e. Construction of the core family - the third-generation family:

[0055] According to the methods in step c and step d, first sort the average breeding values of males and females between families, and then sort the breeding values of individual males and females within families. On the premise of avoiding inbreeding, select the best female individual from the best female family and the best male individual from the best male family to mate, the best male and female individuals from the second-ranked families to mate, and so on, to construct the third-generation core family. And so on, complete all pairings between families to further aggregate excellent genes; during the mating process, avoid mating between male and female individuals from the same family. If the inbreeding coefficient of the paired individuals reaches the set threshold of 1, then mate them with individuals from the next-ranked family respectively;

[0056] f. Continue to construct the core family according to the method in step e for more than 5 generations, and finally obtain a new variety of Pinctada fucata rich in carotenoids.

[0057] In this example, the ASReml software is used to calculate breeding values and heritability parameters based on the BLUP animal model. The analysis model and some codes are as follows:

[0058] !PART 1

[0059] !filter Spacing !select 3

[0060] !continue !maxit 30

[0061] h5 ~ mu Rep !r Fam !f mv

[0062] !PIN !define

[0063] F Add_H 1*4 # 3

[0064] F totalG_H 1+2 # 4

[0065] H herit 3 4 # count heritability

[0066] After running the model, first view the main file fm.asr and find the result part of the variance components. The following operations are as follows:

[0067] Approximate stratum variance decomposition

[0068] Stratum Degrees-Freedom Variance Component Coefficients

[0069] Fam 50.71 10167.1 10.9 1.0

[0070] Residual Variance 500.29 5333.34 0.0 1.0

[0071] Source Model terms Gamma Component Comp / SE % C

[0072] Fam 55 55 0.828737E-01 441.994 2.36 0 P

[0073] Variance 559 551 1.00000 5333.34 15.82 0 P

[0074] The calculation results of heritability are in the fm.pvc file, and the results are as follows:

[0075] 1 Fam 441.994

[0076] 2 Variance 5333.34

[0077] 3 Add_H 1 1768.0 748.72

[0078] 4 totalG_H 1 5775.3 357.71

[0079] herit = Add_H 1 3 / totalG_H 4= 0.3061 0.1239

[0080] Notice: The parameter estimates are followed by their approximatestandard errors.

[0081] Obviously, the above embodiments are merely examples given for clearly illustrating the present invention, rather than limitations on the implementation of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description; it is not necessary and impossible to enumerate all implementation manners here; and the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for cultivating a new variety of Pinctada fucata martensii rich in carotenoids, characterized in that it successively includes the following steps: a. Germplasm resource collection: Collect Pinctada fucata martensii and transport them back to the farm for temporary cultivation until the breeding season; b. Construction of basic families: Dissect the Pinctada fucata martensii collected in step a, measure the carotenoid content of individuals with red, orange-red or light red adductor muscles, and select individuals with high content as parents, and construct full-sib families according to the male-female ratio of 1:1; Each family is cultivated and managed under the same conditions; After the seedlings emerge, they are cultured and managed under the same conditions in the same sea area. When they are cultured for one year, trait measurements are carried out. For each family, more than 1000 male and female individuals are measured; the measured trait indicators include shell length, shell height, and carotenoid content; Then use ASReml software to calculate the breeding value and heritability parameters based on the BLUP animal model; c. Selection of parents: According to the ranking of the family breeding values above, select families higher than the average value as candidate families for seed retention; Rank according to the size of the average breeding value of males and females and number them according to the size. The one with the largest breeding value is numbered 1, and so on; Before the breeding season, conduct individual screening of the selected families by male and female respectively: For each family, select the top 200 male and female individuals according to size for trait measurement and mark each individual, then calculate the breeding value of each individual and rank them. Individuals with a ranking greater than the average value are used as candidate parents respectively; d. Construction of core families - second-generation families: Select the female individual with the first-ranked individual breeding value from the family with the first-ranked female breeding value and the male individual with the first-ranked individual breeding value from the family with the first-ranked male breeding value to mate in a ratio of 1:1; then select the female individual with the first-ranked individual breeding value from the family with the second-ranked female breeding value and the male individual with the first-ranked individual breeding value from the family with the second-ranked male breeding value to mate in a ratio of 1:1; and so on, complete the pairing between all selected families, combine the best with the best, and achieve the purpose of aggregating excellent genes for qualitative traits; The methods of reproduction, seedling cultivation and breeding are the same as those in step b; When cultured for one year, conduct trait measurement and calculation of family breeding value and heritability parameters. The measured trait indicators and the calculation methods of family breeding value and heritability parameters are the same as those in step b; e. Construction of core families - third-generation families: According to the methods of step c and step d, first rank the average breeding values of males and females between families, and then rank the breeding values of males and females within families. On the premise of avoiding inbreeding, select the best female individual from the best female family and the best male individual from the best male family to mate, the best male and female individuals from the second-ranked male and female families to mate, and so on, to construct the third-generation core families, and so on, complete the pairing between all families, and further aggregate excellent genes; during the mating process, avoid mating between male and female individuals of the same family. If the inbreeding coefficient of the paired individuals reaches the set threshold of 1, they are respectively mated with individuals from the next-ranked family; f. Continue to construct core families according to the method of step e until more than 5 generations, and finally obtain a new variety of Pinctada fucata martensii rich in carotenoids.

2. The method for cultivating a new variety of Pinctada fucata rich in carotenoids according to claim 1, characterized in that: in step a, at least more than 2000 parents are collected from at least 3 different sea areas or different farms or different strains, and individuals with regular, complete and disease-free appearances are selected.

3. The method for cultivating a new variety of Pinctada fucata rich in carotenoids according to claim 1, characterized in that: in step b, the seedling breeding of all families is carried out under the same conditions. When the seedlings reach about 1 mm, they are transplanted to the sea area for cage culture, and the stocking density is the same.

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