Method for constructing mussel breeding base population based on three-way hybridization

Abstract

The invention provides a method for constructing a mussel breeding base population based on three-way hybridization of mytilus coruscus, mussels and mytilus coruscus, and belongs to the technical field of marine ecological breeding. The method comprises the following steps: hybridizing a mytilus coruscus population and a mytilus coruscus population, then precisely hybridizing hybrid generations of the mytilus coruscus and the mytilus coruscus population, and polymerizing genetic variation among different geographical populations or varieties through ternary matching hybridization to improve the genetic diversity of a breeding basic population, so as to improve the breeding quality of the mytilus coruscus population and the breeding quality of the mytilus coruscus population. Sufficient genetic variation potential is provided for the breeding plan of the mussels, and the success rate of the breeding plan is improved. According to the method, a breeding base population with high genetic variation diversity is constructed through three-way hybridization, the genetic acquisition possibility and production performance of a mussel breeding plan are improved, the breeding time of a new mussel strain is shortened, the mussel germplasm innovation efficiency is improved, and the method has high practical significance in promoting improved variety of the mussel breeding industry.

Description

Technical Field

[0001] The invention belongs to the technical field of marine ecological aquaculture, and in particular relates to a method for constructing a basic population of mussel breeding based on ternary hybridization of Mediterranean mussel, mussel and thick-shell mussel. Background Art

[0003] Constructing a good basic population is the key to the success of the selective breeding program. The basic population must have relatively good phenotypic traits, rich heritable variation, and stable genetic structure, so that it is possible to tap into considerable genetic gains in subsequent breeding programs and effectively improve production traits. However, so far, there has been no report on the construction of a mussel breeding basic population through the three-way hybridization method. Therefore, proposing and implementing innovations in the construction technology of mussel three-way hybridization basic populations has a strong practical significance for the development of mussel germplasm innovation. Summary of the invention

[0004] The present invention provides a method for constructing a basic population for mussel breeding based on ternary hybridization of Mediterranean mussel, mussel and thick-shell mussel. The method utilizes subspecies and interspecific hybridization to increase the genetic diversity of the breeding population, and constructs a basic population for breeding with high genetic variation diversity through ternary hybridization, thereby improving the genetic acquisition possibility and production performance of the mussel breeding program, shortening the cultivation time of new mussel strains, improving the efficiency of mussel germplasm innovation, and promoting the improvement of mussel breeding industry, which has strong practical significance.

[0005] In order to achieve the above object, the present invention provides a method for constructing a basic population for mussel breeding based on ternary hybridization, comprising the following steps:

[0006] By hybridizing the Mediterranean mussel population and the thick-shelled mussel population, and then using their hybrid offspring, the purple thick-shelled hybrid mussel and the mussel population for precise hybridization, through three-way matching hybridization, the genetic variation between different geographical populations or varieties is aggregated, the genetic diversity of the basic breeding population is improved, and sufficient genetic variation potential is provided for the mussel breeding program, thereby improving the success rate of the breeding program.

[0007] Preferably, the hybridization using the Mediterranean mussel population and the thick-shelled mussel population is specifically as follows:

[0008] By using the unidirectional hybridization between the Mediterranean mussel female parent and the southern thick-shelled mussel male parent, a unidirectional hybrid population, namely the purple thick-shelled hybrid mussel, was constructed with the Mediterranean mussel as the female parent and the thick-shelled mussel as the male parent.

[0009] As a preferred method, the one-way hybrid population with Mediterranean mussel as the female parent and thick-shelled mussel as the male parent is constructed as follows:

[0010] When the gonad development of the parent mussels of Mediterranean mussel and thick-shell mussel is basically synchronized, and the female is orange-yellow and the male is milky white and plump and reaches the IVth stage, the eggs are collected and hybridized by combining shade drying and temperature stimulation;

[0011] Then, high-quality eggs and sperm from more than 100 Mediterranean mussels and thick-shelled mussels were collected by spawning a single mussel in a beaker. The eggs were discarded if contaminated by microscopic inspection. The mixed Mediterranean mussel eggs were fertilized with the sperm of the mixed thick-shelled mussels to construct purple thick-shelled hybrid mussels.

[0012] Preferably, the method for achieving substantially synchronous gonadal development of Mediterranean mussels and thick-shelled mussels is:

[0013] Select more than 500 healthy Mediterranean mussel mothers with a size larger than 5 cm, and artificially ripen the broodstock by raising the temperature by 0.2℃-0.4℃ per day according to the conventional ripening method for bivalve molluscs, and finally raise the temperature to 15℃ and control the temperature for spawning; and

[0014] Select more than 500 healthy thick-shelled mussels with a size larger than 7cm, and artificially ripen the broodstock by raising the temperature by 0.3℃-0.5℃ per day according to the conventional ripening method for bivalve molluscs. Finally, raise the temperature to 15℃ and control the temperature before laying.

[0015] Preferably, the Mediterranean mussels are selected from wild populations in northern China, and the species is Mytilus galloprovincialis after molecular identification. The thick-shelled mussels are selected from wild populations in southern China, and the species is Mytilus coruscus after molecular identification.

[0016] As a preferred method, the precise hybridization of the hybrid offspring of the purple thick hybrid mussel and the mussel population is specifically as follows:

[0017] By using the method of spawning a single mussel in a beaker, high-quality eggs and sperm from more than 100 individuals of purple thick hybrid mussel and mussel were collected respectively, and the eggs were discarded if contaminated through microscopic inspection. The eggs of mixed mussels were fertilized with the sperm of mixed purple thick hybrid mussels, and the eggs of mixed purple thick hybrid mussels were fertilized with the sperm of mixed mussels. Positive and negative ternary hybrid populations were constructed respectively, and then they were cultivated according to the conventional intermediate cultivation and culture methods of Mediterranean mussels, thus constructing a basic population for mussel breeding based on ternary hybridization.

[0018] Preferably, the method for achieving substantially synchronous gonadal development of broodstock of mussels is:

[0019] 300 healthy hybrid scallops and 300 mussel colonies with a size larger than 5 cm were selected, and the conventional ripening method of Mediterranean mussels was used to artificially ripen the broodstock by raising the temperature by 0.2℃-0.4℃ per day, and finally raising the temperature to 15℃.

[0020] Preferably, the mussels are selected from wild populations in southern China, and the species is Mytilus edulis after molecular identification.

[0021] Compared with the prior art, the advantages and positive effects of the present invention are:

[0022] 1. The present invention provides a scientific and efficient method for constructing a basic mussel population, which can introduce more genetic resources in a shorter period of time, effectively aggregate heritable excellent traits, establish a high-quality breeding basic population, improve the success rate of mussel breeding programs, and promote the healthy and sustainable development of the mussel farming industry.

[0023] 2. The method of the present invention uses subspecies and interspecific hybridization to increase the genetic diversity of the breeding population, and constructs a breeding basic population with high genetic variation diversity through ternary hybridization, which improves the current non-standard operation of seedling mixing and inbreeding in the mussel breeding industry, resulting in a decrease in genetic diversity of the breeding population, improves the genetic acquisition possibility and production performance of the mussel breeding program, shortens the cultivation time of new mussel strains by 1-2 years, and improves the efficiency of mussel germplasm innovation, which has a strong practical significance for promoting the improvement of mussel breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of constructing the base population for mussel triad hybridization. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] It is generally believed that there are only three types of mussels in my country, the Mediterranean mussel (Mytilus galloprovincialis), the thick-shelled mussel (Mytilus coruscus) and the green mussel (Perna viridis). According to previous work, only the eggs of the Mediterranean mussel and the sperm of the thick-shelled mussel can be hybridized in one direction. Based on our previous work, through simplified genome sequencing, we found that the southern Mediterranean mussel population sampled is actually the mussel (Mytilus edulis) - another species that looks very similar to the purple mussel. There are also reports of the existence of this mussel in Europe and the overlapping areas with the Mediterranean mussel. The mussel (Mytilus edulis) may have been introduced into my country through other routes and reproduced in some areas in the south. Moreover, the genetic structure of the Mediterranean mussel (Mytilus galloprovincialis) and the mussel (Mytilus edulis) is very different, but they can be fully hybridized. Combined with the one-way hybridization with the thick-shelled mussel, it provides us with ideas for carrying out three-way hybridization to maximize the expansion of genetic diversity and improve the genetic basis of farmed mussels.

[0027] Example 1

[0028] In January of the first year, 500 thick-shelled mussels with good development foundation were selected from Shengsi, Zhejiang as the first-generation hybrid southern group broodstock, and transported to the workshop of Shandong Laizhou Hongjie Seedling Co., Ltd. for ripening. The long-distance transportation method of the broodstock is to put the mussels in an insulated box, wrap the ice bottle with a sponge on the mussels, ensure that the temperature in the box is not higher than 10°C, and seal the insulated box with transparent tape. The mode of transportation is air transportation to ensure that the mussels are out of water for no more than 20 hours. 500 Mediterranean mussels with good development foundation were selected from the mussel breeding area in Rizhao, Shandong as the first-generation hybrid northern group broodstock, and transported to the workshop of Shandong Laizhou Hongjie Seedling Co., Ltd. for ripening. The broodstock are kept in the workshop for about one month, during which fresh diatoms are fed with spirulina, egg yolk and other substitute baits.

[0029] When the gonads of the broodstock are gradually becoming full, the mussel seed collection work begins. 300 broodstock from each group are selected and mixed evenly on the cement floor. After drying in the shade for half an hour, the single mussels are spawned in a beaker. High-quality eggs and a small amount of sperm from more than 100 Mediterranean mussels and thick-shelled mussels are collected respectively. If any eggs are contaminated, they are discarded through microscopic inspection. The mixed thick-shelled mussel sperm fertilizes the mixed Mediterranean mussel eggs, and a one-way hybrid population is constructed with Mediterranean mussels as the female parent and thick-shelled mussels as the male parent.

[0030] The fertilized eggs were placed in 22°C seawater for 25 min. 3The eggs were incubated in a cement pool. After 24 hours, the hatched scallop planktonic larvae were selected with a 300-mesh sieve and then continued to be screened at 25 m 3 Cultivated in cement pools, light controlled at 500-1000 lux, continuous or intermittent aeration, dissolved oxygen not less than 5mg / L, pH 7.9-8.2, COD not higher than 2mg / L. Larvae are mainly fed with golden algae in the early stage, and the larval culture density is no more than 6-8 / mL. In the later stage, the mixture of flat algae and golden algae is mainly used, and the larval density is 4-5 / mL. In the metamorphosis period, diatoms are added appropriately and the larvae's stomach is observed under a microscope every day to determine the number and amount of feed (specifically, in general, feed twice a day, the daily feeding amount is 10,000 cells / ml, observe the number of feed in the larvae's stomach, if the number is sufficient and the color is dark brown, no adjustment is required, if the culture water is clear and the larvae's stomach has light food and color, then the amount or number of feeds should be increased as appropriate, on the contrary, if the stomach has sufficient food and the water color is turbid, then the amount or number of feeds should be reduced as appropriate).

[0031] After 20-25 days of cultivation, the larvae begin to develop eye spots and secondary shells, and attach to the pond wall. When the shell length reaches 1000μm-1500μm, the seedlings are brushed off with a brush and put into a 60-mesh, 60cm long, 40cm wide polyethylene mesh bag. The polyethylene mesh bag is hung in the intermediate cultivation pond for intermediate cultivation, and then hung in the Penglai Sea area in August for the second generation of three-way hybridization operation.

[0032] The data of the grow-out period of the first generation of hybrid mussels are shown in Table 1, where ZZ is the Mediterranean mussel control group, HH is the thick-shelled mussel control group, and ZH is the hybrid group constructed with Mediterranean mussel eggs and thick-shelled mussel sperm. Due to the incompatibility of reverse hybridization, the HZ hybrid combination was not constructed.

[0033] Table 1: The grow-out data of the first generation of hybrid mussels bred by constructing a basic population for mussel breeding through three-way hybridization.

[0034] Experimental groups Shell length(mm) Shell height(mm) Shell width(mm) Full wet weight (mm) ZZ <![CDATA[52.26±3.44 a ]]> <![CDATA[25.22±3.59 a ]]> <![CDATA[27.87±2.52 a ]]> <![CDATA[18.98±6.51 a ]]> HH <![CDATA[55.45±7.43 b ]]> <![CDATA[28.32±4.45 c ]]> <![CDATA[28.56±4.28 b ]]> <![CDATA[22.82±7.15 b ]]> ZH <![CDATA[54.71±4.25 b ]]> <![CDATA[26.22±4.67 b ]]> <![CDATA[29.61±4.55 b ]]> <![CDATA[21.63±6.35 b ]]>

[0035] Note: Different letters in the same column of the same experimental group indicate significant differences (P<0.05).

[0036] From the data in Table 1, we can see that the hybrid group (ZH) constructed by Mediterranean mussel eggs and thick-shelled mussel sperm showed a certain growth advantage over the Mediterranean mussel control group (ZZ), but the growth difference with the thick-shelled mussel was not significant, which preliminarily indicates that the genetic basis and breeding potential of hybrid mussels have been effectively improved.

[0037] In February of the following year, 500 healthy hybrid mussels with a size larger than 5 cm were selected from the Penglai experimental sea area, and 500 healthy mussels with a size larger than 5 cm were selected from the Pingtan sea area in Zhejiang Province. The mussels that passed the molecular identification were sampled and transported to the workshop of Shandong Laizhou Hongjie Seedling Breeding Co., Ltd. for ripening. The long-distance transportation method for the broodstock is to put the mussels in an insulated box, wrap the ice bottle with a sponge on the mussels, ensure that the temperature in the box is not higher than 10°C, and seal the insulated box with transparent tape. The mode of transportation is air transport to ensure that the scallops are out of water for no more than 20 hours. The broodstock are kept in the workshop for about a month, during which they are fed with fresh diatoms supplemented with spirulina, egg yolk and other substitute baits.

[0038] When the gonads of the parent mussels are gradually becoming full, the three-way hybridization of mussels is started. After the mussel parent mussels of the two groups are dried in the shade for half an hour, the individuals with mature and good gonad development are selected and placed in a 1L plastic bowl for individual spawning or spermatogenesis. The mixed sperm of 100 individuals from each of the two groups is collected for standby use. The above-mentioned mixed sperm is used to fertilize the ovulating individuals of different groups, and a total of 100 individuals in each group are successfully fertilized. The fertilized eggs in each group are mixed for standby use. A total of positive and negative three-way hybrid fertilized eggs are obtained, and the amount of fertilized eggs in each group is more than 200 million.

[0039] The positive and negative groups of triple hybrid fertilized eggs were placed in 25m of 22℃ seawater. 3 After 24 hours, the hatched mussel larvae were selected with a 300-mesh sieve and then screened at 25 m 3 Cultivated in cement pools, light controlled at 500-1000 lux, continuous or intermittent aeration, dissolved oxygen not less than 5mg / L, pH 7.9-8.2, COD not higher than 2mg / L. Larvae are mainly fed with golden algae in the early stage, and the larval culture density is no more than 6-8 / mL. In the later stage, the mixture of flat algae and golden algae is mainly used, and the larval density is 4-5 / mL. In the metamorphosis period, diatoms are added appropriately and the larvae's stomach is observed under a microscope every day to determine the number and amount of feed (specifically, feed twice a day under normal circumstances, the daily feeding amount is 10,000 cells / ml, observe the number of feed in the larvae's stomach, if the number is sufficient and the color is dark brown, no adjustment is required, if the culture water is clear and the larvae's stomach has light food and color, then the amount or number of feeds should be increased as appropriate, on the contrary, if the stomach has sufficient food and the water color is turbid, then the amount or number of feeds should be reduced as appropriate).

[0040] After 20-25 days of cultivation, the larvae begin to develop eye spots and secondary shells, and attach to the pond wall. When the shell length reaches 1000μm to 1500μm, the seedlings are brushed off with a brush and placed in a 60-mesh, 60cm long, 40cm wide polyethylene mesh bag. The polyethylene mesh bag is hung in the intermediate cultivation pond for intermediate cultivation, and then hung in the Penglai Sea area in September for cultivation. Thus, a high-quality basic population with rich genetic resources and good growth traits is successfully established, which can be used for subsequent mussel selection breeding programs.

[0041] The shell length data of the larval stage of the cultivated ternary hybrid mussel population are shown in Table 2, among which the control group CC is a self-propagated population of hybrid mussels, the control group DD is a self-propagated population of mussels, the ternary hybrid combination CD is a ternary hybrid population constructed from the eggs of hybrid mussels, and the experimental combination DC is a ternary hybrid population constructed from the eggs of mussels.

[0042] Table 2: Shell length data of the larval stage of the three-way hybrid mussel population cultivated by the method of constructing a basic population for mussel breeding through three-way hybridization.

[0043]

[0044] Note: Different letters in the same column of the same experimental group indicate significant differences (P<0.05).

[0045] As can be seen from Table 2, the three-way hybrid population CD group constructed with hybrid mussel eggs and the three-way hybrid population DC group constructed with mussel (Mytilusedulis) eggs showed certain growth advantages over the self-propagated population, which preliminarily indicated that the genetic basis and breeding potential of the three-way hybrid mussel were effectively improved and could be used as the basic population for breeding programs.

Claims

1. A method for constructing a basic population for mussel breeding based on ternary hybridization, characterized in that: The following steps are involved: By hybridizing the Mediterranean mussel population and the thick-shelled mussel population, and then using their hybrid offspring, the purple thick-shelled hybrid mussel and the mussel population for precise hybridization, through three-way matching hybridization, the genetic variation between different geographical populations or varieties is aggregated, the genetic diversity of the basic breeding population is improved, and sufficient genetic variation potential is provided for the mussel breeding program, thereby improving the success rate of the breeding program.

2. The method according to claim 1, characterized in that The hybridization using the Mediterranean mussel population and the thick-shell mussel population is as follows: By using the unidirectional hybridization between the Mediterranean mussel female parent and the southern thick-shelled mussel male parent, a unidirectional hybrid population, namely the purple thick-shelled hybrid mussel, was constructed with the Mediterranean mussel as the female parent and the thick-shelled mussel as the male parent.

3. The method according to claim 2, characterized in that The specific steps of constructing a one-way hybrid population with Mediterranean mussel as the female parent and thick-shelled mussel as the male parent are as follows: When the gonad development of the parent mussels of Mediterranean mussel and thick-shell mussel is basically synchronized, and the female is orange-yellow and the male is milky white and plump and reaches the IVth stage, the eggs are collected and hybridized by combining shade drying and temperature stimulation; Then, high-quality eggs and sperm from more than 100 Mediterranean mussels and thick-shelled mussels were collected by spawning a single mussel in a beaker. The eggs were discarded if contaminated by microscopic inspection. The mixed Mediterranean mussel eggs were fertilized with the sperm of the mixed thick-shelled mussels to construct purple thick-shelled hybrid mussels.

4. The method according to claim 1, characterized in that The method to achieve the basic synchronization of gonadal development of Mediterranean mussels and thick-shell mussel broodstock is: Select more than 500 healthy Mediterranean mussel mothers with a size larger than 5 cm, and artificially ripen the broodstock by raising the temperature by 0.2℃-0.4℃ per day according to the conventional ripening method for bivalve molluscs, and finally raise the temperature to 15℃ and control the temperature for spawning; as well as Select more than 500 healthy thick-shelled mussels with a size larger than 7cm, and artificially ripen the broodstock by raising the temperature by 0.3℃-0.5℃ per day according to the conventional ripening method for bivalve molluscs. Finally, raise the temperature to 15℃ and control the temperature before laying.

5. The method according to any one of claims 1 to 4, characterized in that: The Mediterranean mussels were selected from wild populations in northern China, and the species was identified as Mytilus galloprovincialis through molecular identification. The thick-shelled mussels were selected from wild populations in southern China, and the species was identified as Mytilus coruscus through molecular identification.

6. The method according to claim 1, characterized in that The specific steps of using the hybrid offspring of purple thick hybrid mussel and mussel population for precise hybridization are as follows: By using the method of spawning a single mussel in a beaker, high-quality eggs and sperm from more than 100 individuals of purple thick hybrid mussel and mussel were collected respectively, and the eggs were discarded if contaminated through microscopic inspection. The eggs of mixed mussels were fertilized with the sperm of mixed purple thick hybrid mussels, and the eggs of mixed purple thick hybrid mussels were fertilized with the sperm of mixed mussels. Positive and negative ternary hybrid populations were constructed respectively, and then they were cultivated according to the conventional intermediate cultivation and culture methods of Mediterranean mussels, thus constructing a basic population for mussel breeding based on ternary hybridization.

7. The method according to claim 6, characterized in that The method for achieving basically synchronous gonadal development of mussel broodstock is: 300 healthy hybrid scallops and 300 mussel colonies with a size larger than 5 cm were selected, and the conventional ripening method of Mediterranean mussels was used to artificially ripen the broodstock by raising the temperature by 0.2℃-0.4℃ per day, and finally raising the temperature to 15℃.

8. The method according to claim 6 or 7, characterized in that: The mussels were selected from wild populations in southern China, and the species was Mytilus edulis after molecular identification.

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