A method for breeding practicable tussah silkworm material with high nutritional value of pupal protein

Abstract

The present invention belongs to the technical field of tussah material cultivation, and specifically discloses a method for breeding tussah materials with high nutritional value of protein in the pupal stage, which is practical. The method includes determining the nutritional value index of protein in the pupal stage, evaluating germplasm resources, selecting and grouping parents, and hybridization breeding. The present invention sets reasonable indicators as a reference to screen for excellent germplasm resource grouping and hybridization, simulates the natural living environment of tussah silkworms in the diapause pupal stage, which is low temperature and cool in winter, through parents and offspring, and applies pressure selection for generations. In response to the phenomenon of increased consumption of protein nutrients in adverse environments, the method comprehensively selects seeds with excellent protein content, cocoon quality, production performance and other indicators, thereby obtaining tussah silkworm materials with good practicality and high protein content in the pupal stage, making the breeding materials more reliable and practical. The new materials created by the present invention can provide a foundation for the breeding and comprehensive utilization of new varieties of tussah silkworms and industrial development.

Description

Technical Field

[0001] The invention relates to the technical field of tussah material cultivation, in particular to a method for breeding practicable tussah materials with high nutritional value of pupal stage protein. Background Art

[0002] The tussah silkworm (Antheraea pernyi) originated in China, where the tussah silkworm industry boasts a history of over 2,000 years. my country is the world's largest tussah silkworm producer, accounting for over 90% of the global tussah silkworm production. Tussah silkworms are a key insect protein food source, boasting a rich nutritional profile. The pupal stage, in particular, represents the peak of nutrient accumulation over a generation, making them highly sought after by the general public. Consequently, consumer demand for edible tussah silkworm pupae has rapidly increased. Breeding tussah silkworms with high-quality pupal protein can address this demand and ensure the nutritional value of tussah silkworm pupae is more scientifically tailored to human health needs. By also taking into account the practicality of the materials, the resulting tussah silkworm breeding materials offer significant economic, social, and ecological benefits.

[0003] Silkworm pupae are the only insect food on the "List of New Food Resources Managed as Ordinary Foods" approved by the Ministry of Health. As a traditional insect food, tussah silkworm pupae have attracted the attention of nutritionists due to their high protein content, balanced nutritional profile, short breeding cycle, and abundant resources. Research has shown that the protein content of tussah silkworm pupae is higher than that of eggs and pork, and is mostly globulin and albumin, making it easily digestible and absorbable by the human body. Tussah silkworm pupae protein contains 18 amino acids, and the content of essential amino acids, except for phenylalanine, is higher than that of pork, beef, and lamb, the three major meats. The amino acid composition is well-proportioned and close to the standard protein and amino acid content patterns recommended by the WHO / FAD, making it an easily absorbed, high-quality protein. As people's living standards improve, the demand for protein is increasing, and edible insect resources such as tussah silkworm pupae have become popular in the market. Chinese patent application No. 201810000517.9 discloses a method for cultivating monovoltine Cordyceps militaris, which includes the steps of strain preparation - culture medium preparation - strain expansion - inoculation - hardening - cultivation - harvesting, etc. The monovoltine organic tussah silkworm Cordyceps militaris cultivated by inoculating Cordyceps fungi onto monovoltine organic tussah silkworm pupae has a high protein content and a variety of amino acids. The cordycepin, cordycepic acid, cordyceps polysaccharide, SOD, and mineral elements such as phosphorus, potassium, zinc, and selenium contained in it can reach or exceed the natural Cordyceps sinensis. The cordycepin content is 3-5 times that of Cordyceps sinensis, and the various bioactive substances are also far higher than those of other fungi. It is an ideal substitute for Cordyceps sinensis.

[0004] However, there are currently no breeding resources targeting high nutritional value of protein in the pupal stage in the research on tussah silkworm breeding. Therefore, under the premise of ensuring practicability, selecting and cultivating tussah silkworm materials with high nutritional value of protein in the pupal stage can not only create a new type of tussah silkworm breeding, but also meet people's health care needs and better promote the quality and efficiency development of the tussah silkworm industry. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for breeding practicable tussah silkworm materials with high nutritional value of pupal stage protein.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] A method for breeding tussah silkworm materials with high nutritional value of pupal protein comprises the following steps:

[0008] S1. Determination of trait indices: Under the same stocking technology and tussah garden conditions, the moisture content of the tussah silkworm during the pupal stage, the fresh pupa protein content, the crude fat content during the pupal stage, the fatty acid content during the pupal stage, the amino acid composition and content during the pupal stage, the trehalose content during the pupal stage, and the glycogen content during the pupal stage were measured; then, according to the conventional protein nutritional value evaluation method, the fresh pupa protein content, the amino acid composition and content during the pupal stage, and the trehalose content during the pupal stage were selected as screening references;

[0009] S2. Germplasm resource evaluation and screening: The fresh pupa protein content, amino acid composition and content during the pupal stage, and trehalose content during the pupal stage of candidate tussah germplasm resources with consistent stocking technology and tussah garden conditions are measured respectively. At the same time, economic traits including yield, vitality, and cocoon quality are taken into consideration as indicators to evaluate and screen candidate tussah germplasm resources;

[0010] S3. Parent selection and pairing: Based on the screening results, select tussah germplasm resources with excellent egg production, practical hatching rate, cocoon collection rate, disease incidence, and cocoon quality as the male parent; select tussah germplasm resources with the relatively highest protein nutritional value index as the female parent, and pair the selected female and male parents into a hybrid combination;

[0011] S4. Artificial pressure selection of parents: The two selected parents are subjected to pressure selection in the natural winter environment during the diapause pupae period to increase the selection pressure and simulate the low temperature and cool living environment of tussah silkworm diapause pupae in the wild in winter. The high-quality areas and individuals after pressure selection are selected;

[0012] S5. Hybrid breeding: Select the optimal areas and individuals with high yield, low disease incidence and excellent cocoon quality traits from the two parents after artificial stress, and hybridize them to obtain the first generation F1. The F1 generation is selected based on egg production and practical hatching rate during the egg stage. Termites are collected in the wild and poor individuals are eliminated. During the silkworm stage, experimental areas with mixed body color and uneven development are eliminated. During the cocoon stage, excellent areas are selected based on cocoon quality traits for continued reproduction;

[0013] S6, systematic separation selection: F1 individuals are self-pollinated to produce the F2 generation, and strict purification selection is carried out during the four metamorphosis stages. Moth areas with mixed body colors are eliminated in the entire area; during the egg stage, areas with fast egg laying, high output rate, and high practical hatching rate are selected for succession and stocking, with 30 moths stocked in a single moth area. During the larval stage, areas with good development uniformity and uniform body color are selected for succession, and cocoons are harvested in the field. During the cocoon stage, the yield, vitality, and cocoon quality traits are statistically analyzed. Based on the protein content of fresh pupae and the trehalose content in the pupal stage, excellent varieties are selected for succession. During the winter pupal diapause period, traditional constant temperature seed preservation is broken, and low temperature and cold pressure are applied to select excellent areas and individuals for succession; the F3-F8 generations repeat the breeding method of the F2 generation, and the F4, F6, and F8 generations are subjected to low temperature and cold pressure, ultimately forming tussah silkworm materials with high nutritional value of pupal protein;

[0014] S7. Material creation and formation: Under the same stocking technology and tussah garden conditions, the F8 generation investigated the protein content of fresh pupae and the trehalose content of pupae in the tussah silkworm materials subjected to low temperature and cold pressure and the tussah silkworm materials stored at a stable 0-2℃ in winter, and verified that low temperature and cold pressure increased the nutritional value of protein in the tussah silkworm pupae; at the same time, the F8-F 13 By selecting for cocoon quality traits from generation to generation, the rate of screening of tussah cocoon layers is increased, and the problem of poor cocoon quality traits caused by high pupa protein content is reduced, thus obtaining practical tussah silkworm materials with high nutritional value of pupal protein.

[0015] Furthermore, in steps S1 and S2, the protein content of fresh tussah pupae is determined by G-250 Coomassie Brilliant Blue colorimetry, the amino acid content and composition of tussah pupae are determined by amino acid analysis, and the trehalose content of tussah pupae is determined by anthrone method.

[0016] Preferably, in step S3, the "Fengwu" tussah germplasm resource is selected as the male parent; and the "Yibao" tussah germplasm resource is selected as the female parent.

[0017] Preferably, in steps S2 and S6, the cocoon yield and cocoon quality trait indicators are the total cocoon quantity, the cocoon layer quantity, the cocoon layer rate, the pupa weight, the number of cocoons collected per moth, and the cocoon collection rate.

[0018] Preferably, in step S7, F8-F 13 The cocoon quality traits of the selected cocoons are total cocoon quantity, cocoon layer quantity, cocoon layer rate and pupa weight.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention screens high-protein nutritional value tussah germplasm resources by setting three indicators: protein content in fresh tussah pupae, amino acid composition and content during the pupal stage, and trehalose content during the pupal stage, providing a highly reliable basis for breeding targets. Simultaneously, the low-temperature, cool living environment of tussah silkworms during their diapause pupal stage is simulated. Starting with parental succession selection, low-temperature, cool living conditions are added during the winter diapause pupal stage. This pressure screening method, aimed at increasing protein nutrient consumption, applies pressure to F2, F4, F6, and F8 generations, achieving higher reliability and targeting for the breeding target. This accelerates the selection of high-protein nutritional tussah materials. Furthermore, by selecting materials with excellent production-practical traits such as cocoon quality, yield, and vitality over multiple generations, the conversion of tussah pupal protein to silk protein is balanced, ensuring the stability, high yield, and practicality of high-protein nutritional tussah materials for field stocking. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the breeding process of the breeding method of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] The tussah germplasm resources used in the following examples are “Yibao”, “Fengwu”, 9906, 9418 and 8821, all of which were obtained from the same tussah garden and the same stocking method in Liaoning Province Sericulture Scientific Research Institute.

[0024] Reference Figure 1 , breeding practicable tussah silkworm materials with high nutritional value of pupal protein, the specific steps are as follows:

[0025] (1) Determination of trait indices: Under the condition of consistent stocking technology and tussah garden conditions, comprehensive indices and parameters of tussah nutritional value are determined. During the pupal stage, the moisture content, fresh pupa protein content, crude fat content, fatty acid content, amino acid composition and content, trehalose content, and glycogen content of tussah are measured. According to the protein nutritional value evaluation method, the fresh pupa protein content is determined among the 7 indices (fresh pupa protein content is the most intuitive indicator, which directly reflects the protein nutritional value evaluation indicators such as protein content, nutrition, and energy value of tussah pupae); amino acid composition and content of tussah pupae (amino acid composition and content can be used to obtain amino acid scores and other data of tussah pupae, which is in line with the method of protein nutritional value evaluation); trehalose content of pupa (trehalose is widely used to preserve protein stability, protects protein under adverse conditions, and indirectly reflects protein content). These three indices are used as references for trait screening.

[0026] In some embodiments, the G-250 Coomassie Brilliant Blue colorimetric method can be used to determine the protein content of fresh tussah pupae, the amino acid analysis method can be used to determine the amino acid content and composition of tussah pupae, and the anthrone method can be used to determine the trehalose content of tussah pupae.

[0027] (2) Evaluation and screening of germplasm resources: The fresh protein content, amino acid composition and content during the pupal stage, and trehalose content during the pupal stage of 15 female and 15 male pupae of 5 candidate tussah germplasm resources, namely, Yibao, Fengwu, 9906, 9418, and 8821, which were consistent with the stocking technology and tussah garden conditions, were measured respectively. At the same time, practical production indicators such as cocoon yield, vitality, and cocoon quality traits were taken into consideration as standards. Among them, practical indicators such as cocoon yield and cocoon quality were the total cocoon quantity, cocoon layer quantity, cocoon layer rate, pupa weight, number of cocoons collected per moth, and cocoon collection rate. The candidate tussah germplasm resources were evaluated and screened.

[0028] (3) Parent selection and combination: Based on the screening results, the “Fengwu” variety with excellent practical traits such as egg production, practical hatching rate, ant collection and cocoon formation rate, disease incidence, and cocoon quality was selected as the male parent; the “Yibao” variety with the relatively highest protein nutritional value index was selected as the female parent, and the selected female and male parents were combined into a hybrid combination.

[0029] (4) Artificial pressure selection of parents: In winter, the diapause pupae of the two parents are subjected to pressure selection in a low-temperature cold environment, breaking the traditional 0-2℃ constant temperature seed storage method. The diapause pupae of the two parents are divided in half, half of which are stored in a site without heating facilities to simulate the survival habits of tussah silkworms in the wild and increase protein consumption, while the other half are kept at a normal constant temperature to ensure material backup. Priority is given to selecting the excellent areas selected by low-temperature cold pressure for hybridization.

[0030] The present embodiment breaks away from the traditional method of preserving seeds in a constant temperature warehouse and utilizes the low temperature and cool natural environment conditions in winter to simulate the wild, undomesticated living environment of tussah silkworms. The adverse environment increases the selection pressure and the consumption of nutrients, and the excellent materials after the pressure is applied are retained, while the weak and inferior species are eliminated.

[0031] (5) Hybrid breeding: The first generation is F1, which is obtained by hybridizing the best areas and individuals with high yield, low disease incidence and excellent cocoon quality traits in "Yibao" and "Fengwu" after artificial pressure. The F1 generation is selected according to the egg production and practical hatching rate during the egg stage. The ants are collected in the wild and the poor individuals are eliminated. The experimental areas with mixed body color and uneven development are eliminated during the silkworm stage. The excellent individuals are selected for succession according to practical traits such as cocoon quality during the cocoon stage.

[0032] (6) Systematic separation selection: F1 individuals are self-pollinated to produce F2 generations. Strict purification selection is carried out during the four metamorphosis stages. For moth areas with mixed body colors, the entire area is eliminated. During the egg stage, areas with fast egg laying, high output rate, and high practical hatching rate are selected for succession. 30 moths are released in a single moth area. During the larval stage, areas with good development uniformity and uniform body color are selected for succession. Cocoons are laid and harvested in the wild. During the cocoon stage, practical traits such as yield, vitality, and cocoon quality are statistically analyzed. Based on the protein nutritional value index, excellent varieties are selected for succession. Winter simulation step (3) method: During the pupal diapause period, the traditional constant temperature seed preservation is broken. Low temperature and cold pressure are applied to select excellent areas and individuals for succession. The F3-F8 generations repeat the breeding method of the F2 generation and the winter pressure selection of F2, F4, F6, and F8, and finally form a practical tussah silkworm material with high pupal protein nutritional value.

[0033] (7) Material creation and formation: Under the same stocking technology and tussah garden conditions, the F8 generation investigated the protein content of fresh pupae and the trehalose content of pupae in the tussah silkworm materials subjected to low temperature and cold pressure and the tussah silkworm materials stored at a stable temperature of 0-2℃ in winter, and verified that the nutritional value of protein in the tussah silkworm pupae was improved by accumulating low temperature and cold pressure in the generations. 13 By selecting and retaining cocoon quality traits over generations, the cocoon layer rate of tussah silkworms is improved through the indicators of total cocoon quantity, cocoon layer quantity and pupa weight, the conversion of tussah silkworm pupa protein and silk protein is balanced, and the problem of poor cocoon quality traits caused by high pupa protein content is reduced, thus creating a new practical tussah silkworm material with high nutritional value of pupal protein.

[0034] In this embodiment, the specific process of investigating the protein content of fresh pupae and the trehalose content of pupae in the F8 generation of the tussah silkworm materials subjected to low-temperature cold stress and the tussah silkworm materials stored at a stable 0-2°C diapause in winter is as follows: by measuring the protein content of fresh pupae and the trehalose content of pupae in 15 female pupae and 15 male pupae in the stress group and the control group, the stability of the high nutritional value of the protein in the stress group after the stress selection is tested. 13 By using the successive generations of screening for cocoon quality traits, the conversion of tussah pupa protein and silk protein can be balanced, reducing the problem of poor cocoon quality traits caused by high pupa protein content, which is not conducive to practical application.

[0035] Through the implementation of the above breeding process, in F 13 The cocoon quality traits selected after the generation are the best in terms of total cocoon quantity, cocoon layer quantity, cocoon layer rate and pupa weight, which is the target product of this embodiment: a practical tussah material with high nutritional value of pupal protein.

[0036] In summary, the present invention uses high-quality and high-yield "Fengwu" as the male parent and "Yibao" with high protein nutritional value as the female parent. After low-temperature cold pressure selection, hybridization is performed to obtain the F1 generation, and the F2 generation is produced by self-pollination of the F1 generation. Individual selection is performed on the isolated population, and after picking the cocoons, statistics are made on practical traits such as yield, vitality, and cocoon quality. According to the protein nutritional value index, excellent varieties are selected and retained for succession. The breeding method of the F2 generation is repeated in the F3-F9 generations, and the winter pressure selection of F2, F4, F6, and F8 is performed. The protein nutritional value index of the low-temperature cold material of the alternate generation is investigated in the F8 generation. At the same time, the F8-F 13 The excellent cocoon quality traits are selected for generations, and finally a practical tussah material with high pupal protein nutritional value is formed. The breeding method described in the present invention can ensure the stable inheritance of the excellent quality of the parents, shorten the breeding period, and reasonably determine the protein nutritional value index, reduce the workload, and improve the reliability of selection; increase the pressure of the low temperature and cool environment, simulate the living state of the tussah silkworm without artificial domestication, and use the adverse environment to increase the consumption of protein, which can speed up the acquisition of tussah silkworm high pupa protein nutritional value materials; at the same time, comprehensively consider the cocoon quality, yield, vitality and other traits of the material, avoid the problem of being unfavorable to practical application due to the improvement of a single breeding goal, and can select new varieties suitable for production needs in a targeted manner.

[0037] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for breeding tussah silkworm materials with high nutritional value of pupal protein, characterized by: The following steps are involved: S1. Determination of trait indices: Under the same stocking technology and tussah garden conditions, the moisture content of the tussah silkworm during the pupal stage, the fresh pupa protein content, the crude fat content during the pupal stage, the fatty acid content during the pupal stage, the amino acid composition and content during the pupal stage, the trehalose content during the pupal stage, and the glycogen content during the pupal stage were measured; then, according to the conventional protein nutritional value evaluation method, the fresh pupa protein content, the amino acid composition and content during the pupal stage, and the trehalose content during the pupal stage were selected as screening references; S2. Germplasm resource evaluation and screening: The fresh pupa protein content, amino acid composition and content during the pupal stage, and trehalose content during the pupal stage of candidate tussah germplasm resources with consistent stocking technology and tussah garden conditions are measured respectively. At the same time, economic traits including yield, vitality, and cocoon quality are taken into consideration as indicators to evaluate and screen candidate tussah germplasm resources; S3. Parent selection and pairing: Based on the screening results, select tussah germplasm resources with excellent egg production, practical hatching rate, cocoon collection rate, disease incidence, and cocoon quality as the male parent; select tussah germplasm resources with the relatively highest protein nutritional value index as the female parent, and pair the selected female and male parents into a hybrid combination; S4. Artificial pressure selection of parents: The two selected parents are subjected to pressure selection in the natural winter environment during the diapause pupae period to increase the selection pressure and simulate the low temperature and cool living environment of tussah silkworm diapause pupae in the wild in winter. The high-quality areas and individuals after pressure selection are selected; S5. Hybrid breeding: Select the optimal areas and individuals with high yield, low disease incidence and excellent cocoon quality traits from the two parents after artificial stress, and hybridize them to obtain the first generation F1. The F1 generation is selected based on egg production and practical hatching rate during the egg stage. Termites are collected in the wild and poor individuals are eliminated. During the silkworm stage, experimental areas with mixed body color and uneven development are eliminated. During the cocoon stage, excellent areas are selected based on cocoon quality traits for continued reproduction; S6, systematic separation selection: F1 individuals are self-pollinated to produce the F2 generation, and strict purification selection is carried out during the four metamorphosis stages. Moth areas with mixed body colors are eliminated in the entire area; during the egg stage, areas with fast egg laying, high output rate, and high practical hatching rate are selected for succession and stocking, with 30 moths stocked in a single moth area. During the larval stage, areas with good development uniformity and uniform body color are selected for succession, and cocoons are harvested in the field. During the cocoon stage, the yield, vitality, and cocoon quality traits are statistically analyzed. Based on the protein content of fresh pupae and the trehalose content in the pupal stage, excellent varieties are selected for succession. During the winter pupal diapause period, traditional constant temperature seed preservation is broken, and low temperature and cold pressure are applied to select excellent areas and individuals for succession; the F3-F8 generations repeat the breeding method of the F2 generation, and the F4, F6, and F8 generations are subjected to low temperature and cold pressure, ultimately forming tussah silkworm materials with high nutritional value of pupal protein; S7. Material creation and formation: Under the same stocking technology and tussah garden conditions, the F8 generation investigated the protein content of fresh pupae and the trehalose content of pupae in the tussah silkworm materials subjected to low temperature and cold pressure and the tussah silkworm materials stored at a stable 0-2℃ in winter, and verified that low temperature and cold pressure increased the nutritional value of protein in the tussah silkworm pupae; at the same time, the F8-F 13 By selecting for cocoon quality traits from generation to generation, the rate of screening of tussah cocoon layers is increased, and the problem of poor cocoon quality traits caused by high pupa protein content is reduced, thus obtaining practical tussah silkworm materials with high nutritional value of pupal protein.

2. The method for breeding tussah silkworm materials with high nutritional value of pupal protein according to claim 1, characterized in that: In the steps S1 and S2, the protein content of fresh tussah pupae is determined by G-250 Coomassie Brilliant Blue colorimetry, the amino acid content and composition of tussah pupae are determined by amino acid analysis, and the trehalose content of tussah pupae is determined by anthrone method.

3. The method for breeding tussah silkworm materials with high nutritional value of pupal protein according to claim 1, characterized in that: In step S3, the "Fengwu" tussah germplasm resource is selected as the male parent; the "Yibao" tussah germplasm resource is selected as the female parent.

4. The method for breeding tussah silkworm materials with high nutritional value of pupal protein according to claim 1, characterized in that: In the steps S2 and S6, the cocoon yield and cocoon quality indicators are the total cocoon quantity, the cocoon layer quantity, the cocoon layer rate, the pupa weight, the number of cocoons collected per moth, and the cocoon collection rate.

5. The method for breeding tussah silkworm materials with high nutritional value of pupal protein according to claim 1, characterized in that: In the step S7, F8-F 13 The cocoon quality traits of the selected cocoons are total cocoon quantity, cocoon layer quantity, cocoon layer rate and pupa weight.

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