BREEDING METHOD OF NEW VARIETY OF HARD-SHELLED RAZOR CLAMS

Abstract

The present invention provides a breeding method of a new variety of hard-shelled razor clams, comprising the following steps: a. Estimating the heritability of shell hardness trait: constructing full-sib families to estimate the heritability of the growth and shell hardness traits of the razor clams; b. Selecting individuals with hard shells from basic population using an electronic hardness tester of material mechanics as the broodstock clams; c. Artificially-induced spawning; d. The offspring grow-out; e. Purification of the new strain: with the new strain of hard-shelled generation 1 adults obtained in step d as broodstocks, repeating the steps c and d several times to obtain a new variety of razor clams with hard shells, and the shell hardness trait of razor clams has stable heritability.

Claims

1. A breeding method of a new variety of hard-shelled razor clams, wherein it comprises the following steps: a. Estimating heritability of shell hardness trait: constructing full-sib families to estimate the heritability of the growth trait and shell hardness trait of the razor clams; b. Parent screening: selecting individuals with hard shells from basic population using an electronic hardness tester of material mechanics as the broodstocks; c. Artificially-induced spawning: the breeding population obtained in step b as broodstocks, washing and sterilizing the parents and then placing them in a well-ventilated position free from sunshine for drying for 2 to 4 hours in the evening, and combining injection of 5-hydroxytryptamine via feet with flowing water stimulation to perform artificially-induced spawning; d. The offspring grow-out: the clams obtained in step c as reproduction population, the rearing of the larvae, spat, adults followed routine bivalve culture procedures to obtain a new strain of razor clams (Generation 1, G1) with high shell hardness; e. Strain purification: the new strain of hard-shelled G1 adults obtained in step d as broodstocks, repeating the steps c and d several times to obtain a new variety of razor clams with hard shells, and the shell hardness trait of razor clams has stable heritability through several generations of consecutive purification.

2. The breeding method of a new variety of hard-shelled razor clams of claim 1, wherein the heritability evaluation in step a is to construct half-sib families using the nested design method, breeding values and heritability for the traits (shell height, shell length, shell width, total weight and shell hardness) of the new variety of hard-shelled clams were calculated.

3. The breeding method of a new variety of hard-shelled razor clams of claim 1, wherein the electronic hardness tester in step b is a mechanical apparatus optimized and applied by the inventor to perform accurate measurement for the shell hardness trait of the razor clams; the specific step is to select 200 individuals and measure the shell hardness using the electronic hardness tester, and to sort the hardness values and determine a constant stress value for the breeding population; the test mode of the electronic hardness tester is modified so that the constant stress value is used to perform parent screening; crushed individuals were eliminated and individuals with high shell hardness were retained as the broodstocks.

4. The breeding method of a new variety of hard-shelled razor clams of claim 1, wherein the step c of injecting 5-hydroxytryptamine is to randomly select 50-100 individuals from the broodstocks and inject 0.1 to 0.2 ml of 5-hydroxytryptamine with 0.02 to 0.04 mmol/L to the feet of the razor clams.

5. The breeding method of a new variety of hard-shelled razor clams of claim 1, wherein in the step e of strain purification process, the several times of repeating the process are preferably 3 to 4 times and the several generations are preferably 2 to 3 generations.

6. The breeding method of a new variety of hard-shelled razor clams of claim 1, wherein the step c of injecting 5-hydroxytryptamine is to randomly select 50-100 individuals from the broodstocks and inject 0.1 to 0.2ml of 5-hydroxytryptamine with 0.02 to 0.04 mmol/L to the feet of the razor clams, and using 0.05mmol/L EDTA solution as a stimulating substance.

Description

EMBODIMENTS

[0017] The technical solutions of the present invention will be further described by referring to the specific embodiments to make these technical solutions clearer and more understandable. Unless otherwise stated, the raw materials used in the embodiments of the present invention are materials commonly used in the field and the methods used in the embodiments are also conventional methods of the field.

1. Family Construction and Heritability Evaluation of the Shell Hardness Trait

[0018] a. Broodstocks screening: on Oct. 1, 2018, a cultured population of S. constricta was collected in Changjie town, Ningbo city, China. The razor clams with shell length of about 6 cm were selected. 100 razor clams with high shell hardness were screened out by using the electronic hardness tester with a pressure of 22 N.Math.mm.

[0019] b. Family construction: After injecting of 5-hydroxytryptamine (0.04 mM) via feet and flowing water stimulation, the individuals were placed into a beaker with a water temperature 20-26° C. and salinity 12-15 ppt for the single spawning induction. 36 hard-shelled full-sib families (comprising 12 half-sib families) were constructed using the method of nested design, while three control group families were constructed.

[0020] c. Larva culture and grow-out: The processes of fertilization and incubation, larva culture, spats nursery and pond culture may be referred to the conventional larvae or spat culture management method; during the entire life cycle, the clam density of each family is consistent to prevent cross contamination. Finally, 27 full-sib families (comprising 9 half-sib families) and 3 control group families were left.

[0021] d. Heritability evaluation: The individual animal model of the ASReml 3.0 software was used to perform variance analysis on the shell hardness traits of the 27 full-sib families at the 10-month-old, the moderate heritability (0.32±0.16) of the shell hardness trait was observed in S. constricta, which implied the genetic improvement for shell hardness can be carried out by selection breeding method. The data of the shell hardness and the growth-related traits is shown in Table 1. Compared with the control groups, the hard-shelled families have average shell hardness, shell length, shell width, shell height and wet weight increased by 15.2%, 12.1%, 10.1%, 8.5% and 44.7%, respectively.

TABLE-US-00001 TABLE 1 Comparison of shell hardness and growth traits between hard-shelled families and control families in S. constricta at the 10-month-old Hard-shelled families Control families Average Standard Variation Average Standard Variation Traits Heritability value difference coefficient value difference coefficient Shell 0.32 ± 0.16 19.62 4.40 22.43 17.03 3.55 20.85 hardness Shell 0.50 ± 0.15 54.07 3.17 5.86 48.22 0.97 2.01 length Shell 0.36 ± 0.13 17.84 1.34 7.51 16.20 0.75 4.63 height Shell 0.56 ± 0.12 12.79 1.07 8.37 11.79 1.07 9.08 width Shell 0.60 ± 0.16 10.13 1.90 18.76 7.00 1.09 15.57 weight

2. Breeding of a New Strain of Hard-Shelled S. constricta

[0022] a. Broodstock screening: On Aug. 20, 2019, a wild population of S. constricta was collected in a coastal intertidal zone of Changjie town (29.19° N, 121.77° E), Ningbo city, China. 70 kg of 1-year-old razor clams with the shell length of about 6 cm were selected, and the shell hardness of 200 individuals were measured by the electronic hardness tester to establish a normal distribution diagram of the trait. Then the broodstocks were screened with constant pressure value of 24 N.Math.mm and a selection intensity of 20%. Finally, a total of 1,120 individuals were retained comprised the G0 broodstock after remove individuals with poor activity.

[0023] b. Maturity acceleration of broodstocks: The disinfected sea mud of the intertidal zone was spread in an indoor cement pond to form a thickness of about 8 cm. 1,120 razor clams obtained in step a were spread uniformly in the pond with water changed one to two times a day. Dead individuals were removed daily. Artificial microalgae Chaetoceros muelleri, Phaeodactylum tricornutum and Tetraselmis sp. were provided for the nutrition enhancement to improve the spawning rate of the wild clams. After enhanced cultivation of 20 d, the gonads of the 1,120 clams were matured.

[0024] c. Offspring grow-out: The 1000 razor clams obtained in the step b comprised the breeding population, spawning induction was performed by combination of injection of 5-hydroxytryptamine (0.04 mM) via feet and flowing water stimulation. After fertilization and incubation, larva cultivation, intermediate rearing and pond culture, a new strain of hard-shelled S. constricta (G1) was obtained.

[0025] d. The strain purification: The steps b to c were repeated to generate the second generation (G2) using the G1 adults as broodstocks on Aug. 25, 2020. Through two consecutive generations of purification, a new strain of S. constricta with hard-shells was obtained. In September of 2020 and 2021, 100 12-month-old razor clams of G1 and G2 were randomly sampled to detect genetic progress, and the shell hardness and growth data were shown in Table 2. After purification of two consecutive generations, the shell hardness trait of S. constricta strain was increased by 30.8% and the growth trait was increased by above 10%, indicating obvious effect of genetic improvement.

[0026] In conclusion, by selecting individuals with high shell hardness of two consecutive generations, a new S. constricta strain with high shell hardness and fast growth traits which is suitable for commercial production is obtained, the work providing important materials for higher production, quality improvement and industrial upgrade of S. constricta.

TABLE-US-00002 TABLE 2 Comparison of shell hardness and growth traits in the strain of hard-shelled S. constricta Shell Shell length Shell width Shell height Wet weight Shell weight Generations hardness (N) (mm) (mm) (mm) (g) (g) G0 22.16 ± 4.87 56.01 ± 4.44 18.25 ± 1.14 12.22 ± 1.28 11.33 ± 2.02 2.01 ± 0.38 G1 24.73 ± 5.44 58.61 ± 4.36 19.00 ± 1.50 13.89 ± 1.20 11.68 ± 2.69 2.32 ± 0.28 G2 29.21 ± 3.07 62.33 ± 4.05 20.87 ± 1.29 14.03 ± 1.02 12.82 ± 2.67 2.72 ± 0.25