Crossbreeding method for disease-resistant silkworms

Abstract

The invention relates to the technical field of biological breeding, and discloses a crossbreeding method of disease-resistant silkworms. The crossbreeding method comprises the following steps: screening crossbreeding parents of the disease-resistant silkworms, stabilizing the characters of the screened disease-resistant parents of Chinese and Japanese silkworms, and hybridizing the disease-resistant parents of the silkworms after the characters are stabilized, so that the stability of the midgut pyosis resistance characters of the silkworms is improved. According to the midgut-pyosis-resistant bombyx mori obtained by the crossbreeding method of the disease-resistant bombyx mori disclosed by the invention, the resistance of the bombyx mori to midgut-pyosis viruses is successfully improved on the basis of ensuring the practicability. In addition, the cross breeding method disclosed by the invention is simple and high in operability, and breeding can be realized only through hybridization.

Description

Technical Field

[0001] This invention relates to the field of biological breeding technology, and more specifically, to a hybridization breeding method for disease-resistant silkworms. Background Technology

[0002] The silkworm, belonging to the Bombyx mori family of the order Lepidoptera, is also known as the domestic silkworm or mulberry silkworm. It is an economically important insect that feeds on mulberry leaves and spins cocoons. The large amount of silk spun by silkworm larvae before pupation, with its high-quality fiber properties and wide range of uses, has become an indispensable raw material for the textile industry, making a significant contribution to the progress of human civilization. To this day, sericulture remains one of the pillar industries in many rural areas, bringing stable income to farmers and promoting agricultural development.

[0003] However, artificially bred silkworms currently face serious disease threats, among which silkworm viral diseases are common in silkworm farming. These mainly include midgut septicemia virus and hematosepticemia virus. Statistics show that the incidence rate of silkworm viral diseases can reach 95%, and the loss rate can reach 15%, which undoubtedly has a significant negative impact on farmers' income, agricultural efficiency, and rural stability. Therefore, the breeding of resistant silkworm viral diseases is particularly important. Summary of the Invention

[0004] The purpose of this invention is to provide a hybrid breeding method for disease-resistant silkworms, which can effectively improve the resistance of silkworms to midgut septicemia virus.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A hybridization breeding method for disease-resistant silkworms includes the following steps: 1) Screening of disease-resistant silkworm hybrid parents yielded Chinese line parent A resistant to midgut septicemia and Japanese line parent B resistant to midgut septicemia; 2) The Chinese strain resistant to midgut septicemia A and the Japanese strain resistant to midgut septicemia B obtained in step 1) were bred to stabilize the disease resistance traits of different strains of silkworms, and to obtain the Chinese strain resistant to midgut septicemia FA1 generation and the Japanese strain resistant to midgut septicemia FB1 generation. 3) Cross the FA1 generation and FB1 generation obtained in step 2) to obtain the F2 generation of silkworms resistant to midgut septicemia; 4) The F2 generation obtained in step 3) is directionally bred to stabilize the disease resistance traits of silkworms after hybridization of different strains, and to obtain the F3 generation of silkworms resistant to midgut septicemia; 5) The F3 generation obtained in step 4) is subjected to crossbreeding in different moth regions to obtain the F4 generation of silkworms resistant to midgut septicemia; 6) Improve the stability of disease resistance traits in the F4 generation obtained in step 5) to obtain the F5 generation of silkworms resistant to midgut septicemia, i.e., the disease-resistant silkworm.

[0006] Further, in step 1), the screening is as follows: feeding artificially bred silkworms with mulberry leaves containing midgut septicemia virus, screening Chinese silkworms without midgut septicemia as Chinese midgut septicemia resistant parent A, and screening Japanese silkworms without midgut septicemia as Japanese midgut septicemia resistant parent B. Furthermore, the artificially bred silkworm is Guican No. 10.

[0007] The purpose of step 1) of this invention is to screen out silkworms with resistance to midgut septicemia from existing artificially bred silkworm populations of different strains, and use them as breeding parents in subsequent breeding processes to introduce relevant genes for subsequent breeding of silkworms resistant to midgut septicemia.

[0008] Further, in step 2), the cultivation is as follows: the Chinese-line resistant to midgut septicemia parent A obtained in step 1) is subjected to multiple hybridizations, single-moth breeding, and fed with mulberry leaves containing midgut septicemia virus until a moth area in which more than 80% of the Chinese-line silkworms are free from midgut septicemia is selected, and the FA1 generation is retained for breeding; the Japanese-line resistant to midgut septicemia parent B obtained in step 1) is subjected to multiple hybridizations, single-moth breeding, and fed with mulberry leaves containing midgut septicemia virus until a moth area in which more than 80% of the Japanese-line silkworms are free from midgut septicemia is selected, and the FB1 generation is retained for breeding; Furthermore, the number of moth zones for single-moth breeding is 50.

[0009] The purpose of step 2) of this invention is to stabilize the resistance to midgut septicemia of the breeding silkworm parent through multiple hybridizations, so that after hybridization of two different strains of silkworm parent, it is easier to obtain silkworms with resistance to midgut septicemia.

[0010] In actual breeding, if step 2) is omitted and the Chinese parent A resistant to midgut septicemia (MSS) and the Japanese parent B resistant to MSS are directly crossed, the number of silkworms with MSS resistance will only be about 15%. However, if step 2) is not omitted and the Chinese parent A resistant to MSS and the Japanese parent B resistant to MSS are crossed to stabilize the trait, the number of silkworms with MSS resistance can reach more than 65%. Therefore, by stabilizing the MSS resistance trait of the breeding silkworm parents through multiple crosses, it is easier to obtain silkworms with MSS resistance.

[0011] The purpose of step 3) of this invention is to obtain silkworms with resistance to midgut septicemia by crossing two different strains of silkworms, generation FA1 and generation FB1, which have stable resistance to midgut septicemia.

[0012] Further, in step 4), the directional breeding is as follows: the F2 generation obtained in step 3) is subjected to multiple hybridizations, single moth breeding, and fed with mulberry leaves containing midgut septicemia virus until the moth area with more than 90% of the silkworms free of midgut septicemia is selected, and the F3 generation is retained for breeding. Furthermore, the number of moth zones for single-moth breeding is 50.

[0013] The purpose of step 4) of this invention is to stabilize the resistance to midgut septicemia in silkworms through multiple hybridizations.

[0014] Further, in step 5), the hybridization of different moth areas is as follows: the male moths in each moth area selected in step 4) are mated with the female moths in other selected moth areas, and single-moth breeding is carried out. Moth areas in which more than 90% of the silkworms are free of midgut pustulosis are selected, and the F4 generation is used for breeding. Furthermore, the number of moth zones for single-moth breeding is 50.

[0015] The purpose of step 5) of this invention is to improve the stability of the resistance to midgut pustulosis in silkworms through crossbreeding in different moth regions. After about 6 to 7 generations of crossbreeding in step 4), the proportion of silkworms without midgut pustulosis in each moth region can be stabilized at over 80%, but the highest proportion can only reach about 95%, indicating the presence of non-homozygous individuals. Although continued crossbreeding can further increase the proportion of homozygous individuals to some extent, the increase is slow. In actual breeding, starting from the 8th generation, after 5 more generations of crossbreeding, the average proportion of silkworms without midgut pustulosis in each moth region only increased by 1.7%, and a high proportion could never be achieved. Moreover, too many crossbreeding times can lead to instability in the practical traits of silkworms, such as total cocoon weight and cocoon layer weight, which is detrimental to the economic value of silkworms. However, crossbreeding in different moth regions not only improves the proportion of homozygous individuals more efficiently, but also improves the stability of the practical traits of silkworms.

[0016] Further, in step 6), the improvement of the stability of the disease resistance trait is achieved by: performing multiple hybridizations on the F4 generation obtained in step 5), raising single moths, feeding them with mulberry leaves containing midgut pustulosis virus, until screening out a moth area where 100% of the silkworms are free of midgut pustulosis, and then retaining the F5 generation for breeding. Furthermore, the number of moth zones for single-moth breeding is 50.

[0017] The purpose of step 6) of this invention is to further improve the stability of the resistance to midgut septicemia in silkworms through multiple hybridizations, thereby achieving the breeding of silkworms resistant to midgut septicemia.

[0018] Furthermore, in step 6), if the stability of the disease resistance trait of silkworms is improved, and if after 8 to 10 generations of hybridization there are still no silkworms without midgut septicemia accounting for 100% of the moth area, step 5), crossbreeding in different moth areas, needs to be repeated.

[0019] When performing step 6) of this invention, if there is no area where 100% of the moths are free of midgut pustulosis, the reason for repeating crossbreeding in different moth areas is as follows: First, too many crossbreeding times will lead to increased instability of practical traits, which is not conducive to economic benefits; second, if, after 8 to 10 generations of crossbreeding, there is still no area where 100% of the moths are free of midgut pustulosis, even if more generations of crossbreeding are carried out, the proportion will not increase significantly. The reason may be that the male or female moths selected in step 5) cannot achieve homozygosity, and even if further crossbreeding is carried out, it is impossible to achieve an area where 100% of the moths are free of midgut pustulosis.

[0020] In addition, if silkworms are found to be suffering from midgut septicemia during the breeding process, the diseased silkworms must be removed immediately, and the breeding environment must be disinfected to prevent the diseased silkworms from being mixed with healthy silkworms and causing infection, which could lead to breeding failure.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention improves the stability of the resistance to midgut septicemia in silkworms by screening resistant hybrid parents, stabilizing the traits of the selected Chinese and Japanese resistant silkworm parents, hybridizing the resistant parents with stabilized traits, and thus obtaining a silkworm with good practical performance and resistance to midgut septicemia.

[0022] Laboratory rearing experiments have shown that the silkworms bred according to this invention, resistant to midgut septicemia, exhibited the following characteristics after 27 days and 8 hours: an average cocooning rate of 97.50% for fourth instar silkworms, an average healthy pupa rate of 93.88% for fourth instar silkworms, an average dead cocoon rate of 2.45%, an average ordinary cocoon rate of 94.59%, an average cocoon-with-one-cocoon rate of 4.14%, an average total cocoon weight of 1.37g, an average cocoon layer weight of 0.43g, an average cocoon layer rate of 31.37%, and an average midgut septicemia-free rate of 99.90%. This demonstrates that the disease-resistant silkworms bred according to this invention possess excellent practical performance and resistance to midgut septicemia. Laboratory breeding experiments have shown that the stability of the practical traits of the silkworms resistant to midgut septicemia bred by this invention is not significantly reduced compared to the Guican No. 10 (breeding parent), while the stability of the resistance to midgut septicemia bred by this invention is significantly improved. Therefore, the silkworms resistant to midgut septicemia bred by this invention have successfully improved the resistance of silkworms to midgut septicemia virus while ensuring practical traits. Regional trials have demonstrated that the total cocoon weight of the silkworm resistant to midgut septicemia bred by this invention can reach 1.31g, the cocoon layer weight can reach 0.28g, the cocoon layer rate can reach 22.05%, and the midgut septicemia-free rate can reach 99.65%. Therefore, the silkworm resistant to midgut septicemia bred by this invention has good practical performance and disease resistance.

[0023] The hybridization breeding method disclosed in this invention is simple and highly operable, and breeding can be achieved simply by hybridization. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In the following embodiments, the method for preparing mulberry leaves containing midgut septicemia virus is as follows: freshly picked healthy mulberry leaves are soaked in a virus preservation solution with a midgut septicemia virus content of 2000 cps / mL for 24 hours to obtain mulberry leaves containing midgut septicemia virus.

[0026] The following embodiments illustrate a hybrid breeding method for disease-resistant silkworms, comprising the following steps: 1) Screening of disease-resistant silkworm hybrid parents: Artificially bred silkworms were fed with mulberry leaves containing midgut septicemia virus. Chinese silkworms without midgut septicemia were selected as Chinese midgut septicemia resistant parent A, and Japanese silkworms without midgut septicemia were selected as Japanese midgut septicemia resistant parent B. Among them, the artificially bred silkworm is Guican No. 10; 2) Stability of disease resistance traits in Chinese and Japanese silkworm lines: The Chinese-line resistant to midgut septicemia parent A obtained in step 1) was subjected to multiple hybridizations and single-moth breeding. The moths were fed mulberry leaves containing midgut septicemia virus until the screening showed that more than 80% of the moth areas were free of midgut septicemia. The FA1 generation was then used for breeding. Among them, the number of moth-breeding areas is 50; The Japanese resistant midgut septicemia parent B obtained in step 1) was subjected to multiple hybridizations and single-moth breeding. The moths were fed mulberry leaves containing midgut septicemia virus until the area where more than 80% of the moths were free of midgut septicemia was selected. The FB1 generation was then used for breeding. Among them, the number of moth-breeding areas is 50; 3) Hybridization of different silkworm strains: The FA1 generation and FB1 generation obtained in step 2) are crossbred, and silkworms without midgut septicemia are screened out and F2 generation is retained for breeding. 4) Stability of disease resistance traits in silkworms: The F2 generation obtained in step 3) was crossbred multiple times, and single moths were raised. They were fed mulberry leaves containing midgut septicemia virus until the area where more than 90% of the moths were free of midgut septicemia was selected for breeding, and the F3 generation was saved for breeding. Among them, the number of moth-breeding areas is 50; 5) Hybridization in different moth regions: In step 4), the male moths in each of the selected moth areas are mated with the female moths in other selected moth areas. Single moth breeding is carried out. Moth areas in which more than 90% of the silkworms are free of midgut pustulosis are selected for breeding, and the F4 generation is retained. Among them, the number of moth-breeding areas is 50; 6) Improved stability of disease resistance traits in silkworms: The F4 generation obtained in step 5) was subjected to multiple crossbreedings and single-moth breeding. The moths were fed with mulberry leaves containing midgut septicemia virus until the area where 100% of the moths were free of midgut septicemia was selected. The F5 generation was then used for breeding. The F5 generation is the disease-resistant silkworm. Among them, the number of moth-breeding areas is 50; In addition, if, after 8 to 10 generations of hybridization, no silkworms without midgut septicemia have appeared in the moth area when the stability of the disease resistance trait of silkworms is improved in step 6), step 5) crossbreeding in different moth areas needs to be repeated.

[0027] Example Hybrid breeding of disease-resistant silkworms 1) Screening of disease-resistant silkworm hybrid parents: The Guican No. 10 silkworm was fed with mulberry leaves containing midgut septicemia virus. Chinese silkworms without midgut septicemia were selected as Chinese midgut septicemia resistant parent A, and Japanese silkworms without midgut septicemia were selected as Japanese midgut septicemia resistant parent B. 2) Stability of disease resistance traits in Chinese and Japanese silkworm lines: Five hybridizations were performed on the Chinese-line resistant to midgut septicemia parent A obtained in step 1). Each moth was raised in 50 plots. The moths were fed with mulberry leaves containing midgut septicemia virus. The plots with more than 80% of the moths free of midgut septicemia were selected and the FA1 generation was used for breeding. The Japanese-type resistant to midgut septicemia parent B obtained in step 1) was hybridized four times, and 50 single moth breeding plots were established. The moths were fed with mulberry leaves containing midgut septicemia virus. The plots with more than 80% of the Chinese-type silkworms free of midgut septicemia were selected and the FB1 generation was retained for breeding. 3) Hybridization of different silkworm strains: The FA1 generation and FB1 generation obtained in step 2) are crossbred, and silkworms without midgut septicemia are screened out and F2 generation is retained for breeding. 4) Stability of disease resistance traits in silkworms: The F2 generation obtained in step 3) was crossbred four times, and 50 individual moth breeding plots were used. Mulberry leaves containing midgut septicemia virus were used as feed, and 29 moth plots with more than 90% of the silkworms free of midgut septicemia were selected and used for F3 breeding. 5) Hybridization in different moth regions: In step 4), the male moths in each of the 29 moth zones selected were mated with the female moths in other selected moth zones. Each moth was raised in 50 zones. The selected moth zones had more than 90% of their silkworms free of midgut pustulosis, and the F4 generation was used for breeding. 6) Improved stability of disease resistance traits in silkworms: The F4 generation obtained in step 5) was crossbred 7 times, and 50 individual moth breeding plots were prepared. The moths were fed with mulberry leaves containing midgut pustulosis virus. The plots with 100% of the moths free of midgut pustulosis virus were selected and the F5 generation was selected for breeding. The F5 generation is the disease-resistant silkworm.

[0028] Effect verification The disease-resistant silkworm eggs obtained in this embodiment of the invention were released from storage for regrowth. The regrowth standard was carried out in spring according to the regrowth standard for dimorphic, high-silk-producing varieties, and in autumn according to the two-stage simplified regrowth standard. Egg pens with uniform egg color and a one-day hatching rate of over 90% were selected for collection. The eggs were then fed according to conventional methods until the cocooning stage. During the cocooning stage, the cocooning equipment should be covered tightly with gauze to prevent mature silkworms from escaping and affecting the survey results. This process was repeated 4 times. During the rearing period, the following were investigated: the silkworm instar rate, the cocooning rate of fourth instar silkworms, the rate of healthy pupae of fourth instar silkworms, the rate of dead cocoons, the rate of ordinary cocoons, the rate of cocoons in the same cocoon, the total cocoon weight, the cocoon layer weight, the cocoon layer ratio, and the rate of septicemia without midgut. The survey results are shown in Table 1. Table 1. Results of disease-resistant silkworm egg rearing (autumn)

[0029] As shown in Table 1, laboratory breeding has proven that the silkworms resistant to midgut septicemia bred in this invention, after 27 days and 08 hours, have the following average cocooning rates: 97.50% for fourth instar silkworms, 93.88% for healthy pupae, 2.45% for dead cocoons, 94.59% for ordinary cocoons, 4.14% for cocoons from the same cocoon, 1.37g for total cocoon weight, 0.43g for cocoon layer weight, 31.37% for cocoon layer ratio, and 99.90% for no midgut septicemia. This indicates that the disease-resistant silkworms bred in this invention have good practical performance and resistance to midgut septicemia. Furthermore, in Table 1, the column for Guican No. 10 shows the survey results of the feeding performance of Guican No. 10 under the same feeding method. Comparing the feeding results of Guican No. 10 and the midgut septicemia-resistant silkworm bred according to this invention, it can be found that the stability of the practical traits of the midgut septicemia-resistant silkworm bred according to this invention is not significantly reduced compared with Guican No. 10 (breeding parent), while the stability of the midgut septicemia-resistant trait is significantly improved. Therefore, the midgut septicemia-resistant silkworm bred according to this invention successfully improves the resistance of silkworms to midgut septicemia virus while ensuring practical traits.

[0030] Regional trials were conducted on the disease-resistant silkworms obtained in the embodiments of the present invention. Specifically, the disease-resistant silkworm eggs obtained in the embodiments of the present invention were fed in regions A, B, C, and D respectively. The feeding results of the regional trials are shown in Table 2. Table 2. Feeding Results in Regional Trials

[0031] As shown in Table 2, regional trials have demonstrated that the total cocoon weight of the silkworm resistant to midgut septicemia bred by this invention can reach 1.31g, the cocoon layer weight can reach 0.28g, the cocoon layer rate can reach 22.05%, and the midgut septicemia-free rate can reach 99.65%. Therefore, the silkworm resistant to midgut septicemia bred by this invention has good practical performance and disease resistance.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A hybrid breeding method for disease-resistant silkworms, characterized in that, Includes the following steps: 1) Screening of disease-resistant silkworm hybrid parents yielded Chinese line parent A resistant to midgut septicemia and Japanese line parent B resistant to midgut septicemia; 2) The Chinese strain resistant to midgut septicemia A and the Japanese strain resistant to midgut septicemia B obtained in step 1) were bred to stabilize the disease resistance traits of different strains of silkworms, and to obtain the Chinese strain resistant to midgut septicemia FA1 generation and the Japanese strain resistant to midgut septicemia FB1 generation. 3) Cross the FA1 generation and FB1 generation obtained in step 2) to obtain the F2 generation of silkworms resistant to midgut septicemia; 4) The F2 generation obtained in step 3) is directionally bred to stabilize the disease resistance traits of silkworms after hybridization of different strains, and to obtain the F3 generation of silkworms resistant to midgut septicemia; 5) The F3 generation obtained in step 4) is subjected to crossbreeding in different moth regions to obtain the F4 generation of silkworms resistant to midgut septicemia; 6) Improve the stability of disease resistance traits in the F4 generation obtained in step 5) to obtain the F5 generation of silkworms resistant to midgut septicemia, i.e., the disease-resistant silkworm.

2. The hybridization breeding method for disease-resistant silkworms according to claim 1, characterized in that, In step 1), the screening is as follows: artificially bred silkworms are fed with mulberry leaves containing midgut septicemia virus, and Chinese silkworms without midgut septicemia are selected as Chinese midgut septicemia resistant parent A, and Japanese silkworms without midgut septicemia are selected as Japanese midgut septicemia resistant parent B.

3. The hybridization breeding method for disease-resistant silkworms according to claim 2, characterized in that, The artificially bred silkworm is Guican No.

10.

4. The hybridization breeding method for disease-resistant silkworms according to claim 1, characterized in that, In step 2), the cultivation process involves: repeatedly hybridizing the Chinese-line resistant to midgut septicemia parent A obtained in step 1), raising single moths, and feeding them with mulberry leaves containing midgut septicemia virus until screening results in a moth area where more than 80% of the Chinese-line silkworms are free from midgut septicemia, and then retaining the FA1 generation for breeding; and repeatedly hybridizing the Japanese-line resistant to midgut septicemia parent B obtained in step 1), raising single moths, and feeding them with mulberry leaves containing midgut septicemia virus until screening results in a moth area where more than 80% of the Japanese-line silkworms are free from midgut septicemia, and then retaining the FB1 generation for breeding.

5. The hybridization breeding method for disease-resistant silkworms according to claim 1, characterized in that, In step 4), the directional breeding is as follows: the F2 generation obtained in step 3) is crossbred multiple times, single moth breeding is carried out, and mulberry leaves containing midgut septicemia virus are used for feeding until the silkworm area with more than 90% of the moths are screened out and F3 generation is retained for breeding.

6. The hybridization breeding method for disease-resistant silkworms according to claim 1, characterized in that, In step 5), the hybridization of different moth areas is as follows: the male moths in each moth area selected in step 4) are mated with the female moths in other selected moth areas, and single-moth breeding is carried out. Moth areas in which more than 90% of the silkworms are free of midgut septicemia are selected, and the F4 generation is used for breeding.

7. The hybridization breeding method for disease-resistant silkworms according to claim 1, characterized in that, In step 6), the improvement of the stability of the disease resistance trait is achieved by: performing multiple hybridizations on the F4 generation obtained in step 5), raising single moths, feeding them with mulberry leaves containing midgut septicemia virus, until screening out a moth area where 100% of the silkworms are free of midgut septicemia, and then retaining the F5 generation for breeding.

8. The hybridization breeding method for disease-resistant silkworms according to claim 1, characterized in that, If, after 8 to 10 generations of hybridization, no 100% of the moth-growing areas still have silkworms without midgut septicemia, then step 5) crossbreeding in different moth-growing areas needs to be repeated when the stability of the disease resistance trait of silkworms is improved in step 6).

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