A method for assisting in the breeding of silkworm varieties by parthenogenesis

Abstract

The present invention relates to the technical field of new silkworm variety breeding, and discloses a method for assisting silkworm variety breeding by parthenogenesis. A material with a high incidence of parthenogenesis is introduced into the target variety, individuals with the target traits or genes are screened and retained as recurrent parents for hybridization, and they are backcrossed with the recurrent parent variety. Then, excellent individuals with the target traits are selected for homozygous fixation by parthenogenesis and homozygous fixation by bisexual reproduction. By utilizing the characteristic that the high incidence of parthenogenesis in the hybrid offspring is not easily lost when using the material with a high incidence of parthenogenesis, through hybridization, backcrossing and self-crossing of the material with a high incidence of parthenogenesis with the target trait variety and the conventional excellent variety, after the backcrossing is completed, the target traits are homozygously fixed by parthenogenesis, so that homozygosity of the target traits can be achieved within fewer generations, and stable homozygous offspring can be obtained, which is expected to shorten the breeding process.

Description

Technical Field

[0001] The present invention relates to the technical field of new silkworm variety breeding, and particularly to a method for assisting silkworm variety breeding by parthenogenesis. Background Art

[0002] Silkworm variety breeding is a process of selecting and cultivating silkworm varieties with excellent characteristics through a series of scientific methods and technical means. Common methods include systematic separation, hybridization, and mutagenesis. With the development of technology, breeding techniques have also made great progress. Molecular marker-assisted selection, gene editing technology, and molecular design breeding technology have also been continuously introduced into silkworm variety breeding. Since its application, parthenogenesis technology, as an unconventional means, has attracted the attention of silkworm breeders due to its unique advantages. Parthenogenesis refers to the process in which female gametes develop into embryos directly through single cell division under gene control or external factor stimulation without fertilization. Therefore, parthenogenesis can quickly homozygously fix genes and traits.

[0003] Scholars from various countries have carried out a large number of studies on the artificial induction of parthenogenesis in silkworms. In particular, Astaurov explored the temperature and time parameters for heat-stimulated induction of parthenogenesis in silkworms, effectively improving the incidence and hatching rate of parthenogenesis. Wang Yongqiang, Du Xin, Weng Hongbiao, Song Rengsu, etc. further optimized the induction method of parthenogenesis, invented corresponding egg-taking equipment, and created and conserved a large number of materials with a high incidence of parthenogenesis, providing a strong guarantee for the role of parthenogenesis in silkworm variety breeding.

[0004] Patent CN202210785501.X discloses a method for breeding silkworm varieties resistant to septicemia. It introduces disease-resistant genes into the target variety, selects and retains disease-resistant genes by adding toxins in each generation, and backcrosses with the target variety to retain the economic traits of the target variety. The method of mating one male with two females in a numbered manner is used to detect the homozygosity of disease-resistant genes. Similar methods have a small application range, are only used for introducing disease-resistant genes into the target variety, and it is difficult to homozygously pure the disease-resistant genes of the female parent. If the disease-resistant genes of the female parent are not homozygous in the B2 generation, it is necessary to raise single moths from the egg circles with homozygous disease-resistant genes of the male parent in the B3 generation, and perform male-female co-detection cyclically until the disease-resistant genes of the female parent are homozygous. There are disadvantages such as uncertainty, large workload, and long breeding time.

[0005] In the face of the above technical gaps, it is urgent to further study the method of assisting variety breeding by parthenogenesis, find a new method for silkworm variety breeding, simplify experimental design and technical operations, accelerate the fixation of target genes, avoid the accumulation of unfavorable genes or the loss of beneficial genes, and shorten the breeding cycle. Summary of the Invention

[0006] To solve the above-mentioned existing problems, the present invention discloses a method for assisting in the breeding of silkworm varieties by parthenogenesis, which solves the problems that the current breeding methods may involve complex experimental designs and technical operations, resulting in high costs, long breeding cycles, and the accumulation of certain adverse genes or the loss of beneficial genes due to excessive selection or homozygosity of materials over a long period of time.

[0007] The present invention provides a method for assisting in the breeding of silkworm varieties by parthenogenesis. High-incidence parthenogenesis materials are introduced into the target variety, individuals with target traits or genes are screened and retained for crossing with the recurrent parent, backcrossed with the recurrent parent variety, and excellent individuals with target traits are selected for homozygous fixation by parthenogenesis and homozygous fixation by bisexual reproduction. The specific steps are as follows:

[0008] Step 1: Cross a high-incidence parthenogenesis material with the target variety and screen and retain individuals with target traits.

[0009] Step 2: Cross the individuals with target traits obtained in Step 1 with the recurrent parent to obtain the F1 generation.

[0010] Step 3: Backcross the F1 generation obtained in Step 2 with the recurrent parent N times. For each backcross generation, screen for target traits, and select and retain individuals with target traits and excellent characteristics of the recurrent parent for subculture to obtain the introgressed material BCN generation.

[0011] Step 4: Self-cross the BCN generation obtained in Step 3 to obtain the B1 generation, and screen and retain individuals with target traits and excellent characteristics of the recurrent parent.

[0012] Step 5: Simultaneously perform parthenogenesis and testcross on the B1 generation obtained in Step 4. According to the testcross results, select and retain B2 generation parthenogenetic individuals with target traits and excellent characteristics of the recurrent parent for subculture to establish a pure parthenogenetic line.

[0013] The high-incidence parthenogenesis material is a silkworm variety with a high incidence of parthenogenesis, and the characteristic of high incidence of parthenogenesis is not easily lost in the offspring, which comes from the inventor's previous research. Specifically, the parents of the "Zhefeng series" single-cross varieties bred by the Zhejiang Academy of Agricultural Sciences have this characteristic, such as female 29 in Zhefeng No. 1, female 35 in Zhefeng No. 2, and female 35CN in Zhefeng No. 3; other silkworm varieties with a high incidence of parthenogenesis are not excluded here, because in principle, any silkworm variety containing genes related to high incidence of parthenogenesis can be used to assist in variety breeding according to the technical scheme of the present invention.

[0014] The target variety refers to the silkworm variety containing the target trait. Generally speaking, the target trait is a trait that is detectable, easy to judge, heritable, and relatively stable, such as disease resistance, fluorine tolerance, markings, blood color, etc. The trait can be a dominant trait controlled by a single gene, multiple genes without a major gene, or multiple genes with a major gene. It can be a single trait controlled by a single gene or multiple traits controlled by multiple genes, and does not include traits significantly affected by environmental factors and epigenetic inheritance. Similarly, other target traits and corresponding target varieties are not excluded here, because in principle, different target traits do not affect the occurrence of parthenogenesis.

[0015] Backcross (the hybrid produced by crossing two parents is crossed with one of the parents again) generally selects the variety with excellent characteristics as the female parent in the first cross, and as the male parent in subsequent backcrosses. This parent is called the recurrent parent in backcross. In the present invention, to avoid overly complex description, all involved hybrid parents are referred to as recurrent parents.

[0016] Step 5 proposes to conduct parthenogenesis and test cross simultaneously, making full use of the fact that there are still residual eggs in the female moth's body after oviposition stimulated by temperature change. Based on this, test cross is carried out, which can quickly screen whether the target trait of the female parent is homozygous; in most cases, a homozygous parthenogenetic line can be directly obtained, and the probability that all subsequent individuals are heterozygous is very low. A homozygous parthenogenetic line can also be obtained by repeating Step 4 and Step 5 once. Generally speaking, parthenogenesis assisted until the B2 generation can homozygose the target gene, while the conventional method requires after the B3 generation, and the success rate of parthenogenesis assistance is significantly improved.

[0017] Regarding the success rate of the above homozygosity, in the theoretical state, in this method, for a single target trait, at the B2 generation, the success rate of the homozygous parthenogenetic line is 1 / 2, and at the B3 generation, the success rate of the homozygous bisexual reproductive line is 1 / 2; for two target traits, at the B2 generation, the success rate of the homozygous parthenogenetic line is 1 / 4, and at the B3 generation, the success rate of the homozygous bisexual reproductive line is 1 / 4, and so on. For other methods, for a single target trait, at the B3 generation, the success rate of the homozygous bisexual reproductive line is 1 / 4; for two target traits, at the B3 generation, the success rate of the homozygous bisexual reproductive line is 1 / 16, and so on.

[0018] In some ways, Step 5 further includes:

[0019] Step 51: If there is no single-moth egg circle in the B2 generation with homozygous target trait in Step 5, repeat Step 4 and Step 5 until offspring with homozygous target trait and retaining the excellent characteristics of the recurrent parent appear.

[0020] The main reason for repeating Step 51 is that all subsequent individuals may be heterozygous, although this possibility is very small; a homozygous parthenogenetic line can be obtained by repeating parthenogenesis and test cross once.

[0021] In some ways, it also includes the steps:

[0022] Step 61: Use the B1 generation described in Step 4 for self-crossing to obtain a B2 generation self-crossing population;

[0023] Step 62: Screen male individuals in the B2 generation self-crossing population that retain the target traits and the excellent characteristics of the recurrent parent, and configure the B3 generation and test-cross materials in the way of mating one male with two females in a numbered manner. The female parent of the B3 generation bisexual germplasm line is the homozygous parthenogenetic individual obtained in Step 5;

[0024] Step 63: Select B3 generation individuals with the target traits and the excellent characteristics of the recurrent parent according to the test-cross results for subculture to establish a homozygous bisexual germplasm line.

[0025] Adopting the technical solution of the present invention, parthenogenesis and female individual test-cross are carried out in the B1 generation where homozygous individuals appear to quickly homozygose female individuals. If it is necessary to establish a bisexual germplasm line, only the male individuals need to be test-crossed in the B2 generation, without complex experimental designs and technical operations, which can, to a certain extent, avoid the accumulation of non-expected genes in the breeding materials, ensure that excellent traits are not lost, reduce scientific research costs, and shorten the breeding process.

[0026] In some ways, it also includes the steps:

[0027] Step 64: If there is no B3 generation egg circle single moth with homozygous target traits in Step 62, repeat Step 4 and Steps 61 - 62 until offspring with homozygous target traits and the retention of the excellent characteristics of the recurrent parent appear.

[0028] Similarly, the main reason for repeating Step 64 is that the subculture individuals may all be heterozygotes, although this possibility is very small; a homozygous bisexual germplasm line can be obtained by repeating parthenogenesis and test-cross once.

[0029] One of the core applications of the present invention lies in conveniently introducing multiple different target traits into new silkworm varieties: obtaining a B1 generation parthenogenetic line containing multiple target trait genes through hybridization, backcrossing, self-crossing, etc. of multi-target varieties, and verifying whether the multiple target trait genes of the B1 generation parthenogenetic line are homozygous through one test-cross; when the constructed parthenogenetic line is verified to be homozygous for multiple target trait genes through test-cross, it can then carry out bisexual reproduction with the male of the B2 generation self-crossing population, and simultaneously carry out synchronous test-cross on the male of the bisexual reproduction population, and construct a bisexual germplasm line homozygous for multiple target traits according to the test-cross results.

[0030] When it comes to the pyramiding breeding of multiple target traits, conventional breeding methods require multiple test crosses in each generation to verify the homozygosity of the target trait genes of the male or female parent for a certain target trait each time. Therefore, the number of test crosses increases exponentially with the increase in the number of target traits, which is one of the difficulties in current pyramiding breeding. However, when the method of the present invention is adopted, due to the stable genetic property of the parthenogenetic line, only two test crosses are needed to obtain a homozygous bisexual reproductive line. Compared with the conventional breeding method, the method of the present invention has significant advantages in pyramiding breeding and significantly shortens the breeding cycle.

[0031] In some embodiments, the parthenogenetic high-incidence material has a parthenogenetic incidence rate ≥ 90%, a hatching rate ≥ 85%, is stably heritable, and is relatively dominant over the low incidence rate.

[0032] The meaning of "relatively dominant over the low incidence rate" is that the parthenogenetic incidence rate is a quantitative trait, and the dominant and recessive relationships between high and low incidence rates are not absolute, but may show dynamic changes due to gene interaction, environmental conditions, or observation depth.

[0033] In some embodiments, the parthenogenetic high-incidence material includes one or more of female 29 in Zhefeng No. 1, female 35 in Zhefeng No. 2, and female 35CN in Zhefeng No. 3.

[0034] In some embodiments, the target trait is a detectable, easily determinable, heritable, and relatively stable trait, and the trait includes dominant traits controlled by single genes, multiple genes without major genes, and multiple genes with major genes. The trait includes single traits controlled by a single gene and multiple traits controlled by multiple genes.

[0035] In some embodiments, the recurrent parent can be a conventional material (non-parthenogenetic high-incidence material) or a parthenogenetic high-incidence material.

[0036] In some embodiments, the recurrent parent can be a single parent or multiple parents.

[0037] In some embodiments, the number of backcrosses N ≥ 3.

[0038] One of the benefits of appropriately increasing the number of backcrossing is to restore the excellent characteristics of the recurrent parent, introduce the specific excellent genes of the non-recurrent parent into the genetic background of the recurrent parent, improve the homozygosity of the target gene, enable the target trait to be stably inherited, reduce the phenomenon of trait separation, and make the selected varieties more consistent in the performance of the target trait; of course, the more backcrossing times are not necessarily better. One of the disadvantages of too many backcrossing times is linkage drag. During the backcrossing process, some unfavorable genes linked to the target gene may be introduced into the genetic background of the recurrent parent along with the target gene. If the number of backcrossings is too many, the problem of linkage drag may become more serious, resulting in poor performance of certain traits in the offspring and affecting the overall quality of the variety; too many backcrossings will also make the genetic background of the offspring too biased towards the recurrent parent, and the genetic basis will become narrow, which will lead to a decrease in the variety's ability to adapt to environmental changes and make it vulnerable to new diseases and pests or adverse environmental factors. In addition, in subsequent breeding improvements, due to insufficient genetic diversity, it will be more difficult to further improve the comprehensive traits of the variety. Therefore, in hybrid breeding, it is necessary to comprehensively consider various factors, weigh the pros and cons, and determine the appropriate number of backcrossings to achieve the best breeding results.

[0039] Preferably, the number of backcrossings N = 3. The present invention uses a variety of different high-incidence materials of parthenogenesis and different target varieties for variety breeding, and finds that generally speaking, the parthenogenetic and hermaphroditic silkworms finally established after three backcrossings have the best consistency of target traits, comprehensive traits, etc.

[0040] In some embodiments, the genetic composition of the offspring after backcrossing N times contains ≤3.12% of the genetic composition of the material with a high incidence of parthenogenesis.

[0041] During hybrid breeding, the genetic composition of A or B in the offspring of A×B is 1 / 2. After backcrossing B once, A drops to 1 / 4... In order to dilute the genetic composition of the auxiliary variety, multiple backcrossings are required. After three backcrossings, the genetic composition of the auxiliary variety drops to 3.12%; the genetic composition of the backcross offspring is calculated as follows: in the backcross offspring, gametes from F1 account for 1 / 2, and gametes from parent B account for 1 / 2; the genetic composition of gametes from F1 accounts for 1 / 2, and all gametes from parent B contain the genetic composition of B (accounting for 1); then the genetic composition of B in the backcross offspring is 1 / 2×1 / 2+1 / 2×1=1 / 4+1 / 2=3 / 4, and that of A is 1 / 4; and so on, after three backcrossings it is 1 / 32.

[0042] In some embodiments, if the recurrent parent is a high-incidence parthenogenesis material, the genetic composition of the recurrent parent is ≥ 96.88%.

[0043] In summary, the present invention includes at least one of the following beneficial technical effects:

[0044] 1. By taking advantage of the characteristic that the high incidence of parthenogenesis in materials with a high incidence of parthenogenesis is not easily lost in hybrid offspring, through hybridization, backcrossing, and self-crossing of materials with a high incidence of parthenogenesis with varieties with target traits and conventional excellent varieties, and after completing the backcrossing, performing parthenogenetic homozygosity of the target traits, homozygosity of the target traits can be achieved within fewer generations, and stable homozygous offspring can be obtained, which is expected to shorten the breeding process;

[0045] 2. Make full use of the fact that there are still residual eggs in the female moths after egg-laying under variable temperature stimulation. Based on this, synchronous parthenogenesis and test-crossing are carried out, which can quickly screen whether the target traits of the female parent are homozygous; in most cases, a homozygous parthenogenetic line can be directly obtained. In the very low probability case where all the successive individuals are heterozygotes, a homozygous parthenogenetic line can also be obtained by repeating parthenogenesis and test-crossing once; generally speaking, parthenogenetic assistance to the B2 generation can homozygose the target gene, while the conventional method requires after the B3 generation, and the success rate of parthenogenetic assistance is significantly improved;

[0046] 3. If a bisexual reproductive line needs to be established, only the male individuals need to be test-crossed in the B2 generation, without complex experimental designs and technical operations, which can avoid the accumulation of non-expected genes in the selected breeding materials to a certain extent, ensure that excellent traits are not lost, reduce scientific research costs, and shorten the breeding process;

[0047] 4. When it comes to the pyramiding breeding of multiple target traits, the conventional breeding method requires multiple test-crosses in each generation to verify the homozygosity of the target trait genes of the male or female parent for a certain target trait each time. Therefore, the number of test-crosses increases exponentially with the increase in the number of target traits, which is one of the difficulties in current pyramiding breeding; however, when the method of the present invention is adopted, due to the stable genetic characteristics of the parthenogenetic line, only two test-crosses are required to obtain a homozygous bisexual reproductive line: first, parthenogenesis and test-crossing are carried out simultaneously on the parthenogenetic line in the B1 generation. According to the test-cross results, a homozygous parthenogenetic line can be constructed. The homozygous parthenogenetic line and the male individuals in the B2 generation perform bisexual reproduction, and at the same time, the male individuals in the B2 generation are test-crossed. According to the test-cross results, a homozygous bisexual reproductive line can be constructed; compared with the conventional breeding method, the method of the present invention has significant advantages in pyramiding breeding and significantly shortens the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Flow chart of the method for selecting and breeding silkworm varieties assisted by parthenogenesis of the present invention DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention will be further elaborated in detail below in combination with specific embodiments and the accompanying drawings of the specification. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The materials, reagents, etc. used in the following embodiments are commercially available reagents and materials unless otherwise specified.

[0050] The flow chart of the method for utilizing parthenogenesis to assist silkworm variety selection of the present invention is as follows Figure 1 As shown, the method for parthenogenetic assisted silkworm breeding is described in detail below in combination with Examples 1-2.

[0051] Example 1

[0052] Taking resistance to blood-type pus disease of silkworm as the target trait, the disease-resistant variety Qiufeng N was selected as the target variety, and the high-incidence material of parthenogenesis, He 35 (the high-incidence material of parthenogenesis and the recurrent parent are the same material) were bred. The specific breeding method is as follows:

[0053] In the summer of 2015, Qiufeng N with disease resistance gene was crossed with female 35, a material with high incidence of parthenogenesis, to obtain F1 generation. After immediate acid soaking, a high temperature and high humidity environment was set in the autumn of 2015. The F1 generation of ant silkworms was treated with 1.5×10 7 Feed the individuals / ML of Bombyx mori blood-type pus disease polyhedron suspension with mulberry leaves for 24 hours, and select the superior individuals for succession;

[0054] The BC4 generation was obtained by backcrossing the F1 generation and the female 35 four times, and a high temperature and high humidity environment was set for each backcross. 7 Feed the individuals / ML of Bombyx mori blood-type pus disease polyhedron suspension with mulberry leaves for 24 hours, and select the superior individuals for succession;

[0055] In the spring of 2017, after feeding the BC4 generation with poison, high-quality and highly resistant individuals were selected for self-pollination to obtain the B1 generation;

[0056] In the autumn of 2017, the B1 generation was fed with poison in a high temperature and humid environment, and excellent individuals were selected and raised until they emerged. 25 female moths with excellent individuals were selected and marked one by one, and these 25 female moths were simultaneously subjected to parthenogenesis and test cross. The parthenogenesis materials were numbered 1 to 25, and the test cross materials were numbered C1 to C25. The parthenogenesis method was that the virgin moths were placed under natural light for 3 hours, and then placed in a 5℃ refrigerator for 4 days without mating, and then taken out, and then laid eggs in a high temperature environment of 30℃. After 6 hours, the eggs laid by variable temperature stimulation were treated in 46℃ warm water for 18 minutes, and then placed in room temperature water for 5 minutes, taken out and dried, and placed in a biochemical incubator at 16℃ and relative humidity of 80% for 3 days, and then conventional silkworm seed treatment was carried out. For test cross, the female moths that laid eggs under variable temperature stimulation mated with the test cross materials and laid eggs again, and the test cross experiment was carried out using the residual eggs in the female moths that laid eggs under variable temperature stimulation.

[0057] In the spring of 2018, under a hot and humid environment, the B2 generation parthenogenetic offspring and the test cross offspring were treated with a 1.5×10 7The mulberry leaves were fed with a suspension of polyhedra of Bombyx mori blood type nuclear polyhedrosis virus at a concentration of

[0058] The construction of a parthenogenetic line homozygous for the gene resistant to Bombyx mori blood type nuclear polyhedrosis virus is also a type of pyramiding breeding. Specifically, the traits of high parthenogenetic incidence of female 35 and the disease resistance trait of Qiufeng N were simultaneously introduced into a new Bombyx mori variety. Based on the same principle, more different target traits can also be introduced into a new Bombyx mori variety. Especially when there are a large number of target traits, through hybridization, backcrossing, self-crossing, etc. of varieties with multiple target traits and materials with high parthenogenetic incidence, a parthenogenetic line of the B1 generation containing genes for multiple target traits can be obtained. By means of a single test cross, it can be verified whether the genes for multiple target traits in the parthenogenetic line of the B1 generation are homozygous, and the corresponding parthenogenetic individuals of the B2 generation can be selected to construct a parthenogenetic line homozygous for genes of multiple target traits.

[0059] Example 2

[0060] Take the B1 generation in Example 1, select excellent individuals for self-crossing after feeding with virus, and obtain a self-crossing population of the B2 generation. Select 25 excellent male moths, one male mates with two females, where one female is the parthenogenetic offspring of the B2 generation in Example 1, and the other female is a female of the test cross material. Under a high-temperature and high-humidity environment, the newly hatched silkworm larvae of the self-crossing population of the B2 generation and the offspring of the test cross material were fed with mulberry leaves coated with a suspension of polyhedra of Bombyx mori blood type nuclear polyhedrosis virus at a concentration of 1.5×10 7 per milliliter for 24 hours to conduct the test for homozygosity of the disease resistance gene in the offspring of bisexual reproduction. After testing, all the individuals in a total of 10 egg circles (moth areas) of the test cross offspring survived, indicating that the male moths corresponding to the 10 egg circles were homozygous for the disease resistance gene. Selecting the corresponding bisexual reproductive offspring of the B3 generation can construct a bisexual reproductive line homozygous for the gene resistant to Bombyx mori blood type nuclear polyhedrosis virus.

[0061] Based on the same principle, a bisexual reproductive line homozygous for genes of multiple target traits can be constructed. When the parthenogenetic line constructed has been verified by test cross to be homozygous for genes of multiple target traits, it can then be used for bisexual reproduction and synchronous test cross with the males in the self-crossing population of the B2 generation in the way of one male mating with two females, and a bisexual reproductive line homozygous for multiple target traits can be constructed according to the test cross results.

[0062] Comparative Example 1

[0063] The target variety and cultivation method are the same as in Example 1, except that instead of using the parthenogenesis high-incidence material of the present invention, a conventional material (non-parthenogenesis high-incidence material) is used. In the spring of 2017, 40 female moths and 40 male moths with excellent individuals were selected from the B1 generation, and these 40 male moths were marked one by one with numbers 1 to 40. The B2 generation for self-crossing and seed preservation and the test-cross materials were prepared by the method of mating one male with two females in a numbered manner, and the eggs laid by the female moths were stored refrigerated. The male moths after self-crossing were then test-crossed with the female moths of the virus-sensitive variety Kasuga B. The male moths for self-crossing and seed preservation corresponded one by one with those for test-crossing. In the summer of 2017, the male parent disease-resistant gene homozygosity of the test-cross materials was detected. After detection, all individuals in a total of 5 egg circles (moth areas) survived. The corresponding self-crossed B2 generation egg circles with 5 were male parent disease-resistant gene homozygous egg circles; in the mid-autumn of 2017, the male parent disease-resistant gene homozygous egg circles were individually fed with virus until they emerged as adults, and 40 excellent female and male moths were respectively selected from each moth area, and the female and male moths were numbered respectively. The B3 generation for self-crossing and seed preservation and the test-cross materials were prepared by the method of mating one male with two females in a numbered manner. Specifically, the male moths were separated after self-crossing with the female moths in the same moth area, that is, the same egg circle. After being numbered, the self-crossed B3 generation was stored refrigerated; the male moths after self-crossing were then test-crossed with the female moths of the virus-sensitive variety, and the egg circles laid by the female moths after test-crossing were numbered one by one. In the late autumn of 2017, the female parent and male parent disease-resistant gene homozygosity of the test-cross materials were detected, that is, both male and female were tested. All individuals in all egg circles (moth areas) survived. The corresponding B2 generation moth areas were female parent disease-resistant gene homozygous (the male parent disease-resistant gene was homozygous in the previous round of detection). If all individuals in a certain egg circle (moth area) survived, it was the male parent disease-resistant gene homozygous of the corresponding B3 generation egg circle.

[0064] Compared with Comparative Example 1, the advantages of Examples 1-2 are as follows: (1) The process of variety breeding is shortened. With the assistance of parthenogenesis to the B2 generation, the target gene can be homozygous, while the method adopted in Comparative Example 1 requires after the B3 generation; (2) The success rate of parthenogenesis assistance is significantly improved; (3) The residual eggs in the female moths after egg-laying stimulated by variable temperature are fully utilized. Based on this, synchronous parthenogenesis and test-cross are carried out, which can quickly detect whether the target traits of the female parent are homozygous; (4) It is not limited to parthenogenetic lines. According to needs, bisexual reproductive lines can also be established. Only the male individuals need to be test-crossed in the B2 generation, without complex experimental designs and technical operations, which can avoid the accumulation of non-expected genes in the breeding materials to a certain extent and ensure that excellent traits are not lost.

[0065] In addition, when it comes to the pyramiding breeding of multiple target traits, conventional breeding methods require multiple test crosses in each generation to verify the homozygosity of the target trait genes of the male or female parent for a certain target trait each time. Therefore, the number of test crosses increases exponentially with the increase in the number of target traits, which is one of the difficulties in current pyramiding breeding. However, when the method of the present invention is adopted, due to the stable genetic properties of the parthenogenetic line, only two test crosses are needed to obtain a homozygous amphimictic line: First, the parthenogenetic line of the B1 generation is subjected to parthenogenesis and test cross simultaneously. According to the test cross results, a homozygous parthenogenetic line can be constructed. The homozygous parthenogenetic line and the male individuals of the B2 generation perform amphimixis, and at the same time, the male individuals of the B2 generation are subjected to test cross. According to the test cross results, a homozygous amphimictic line can be constructed. Compared with the conventional breeding method, the method of the present invention has significant advantages in pyramiding breeding and significantly shortens the breeding cycle.

[0066] As described above, the above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any equivalent modifications or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for assisting the breeding of silkworm varieties by parthenogenesis, characterized in that, Including the steps: Step 1: Hybridize the parthenogenesis high-incidence material with the target variety, and screen and retain the individuals with the target traits; the parthenogenesis incidence of the parthenogenesis high-incidence material is ≥90%, the hatching rate is ≥85%, and it is stably inherited and relatively dominant to the low incidence. Step 2: Hybridize the individuals with the target traits obtained in Step 1 with the recurrent parent to obtain the F1 generation. Step 3: Backcross the F1 generation obtained in Step 2 with the recurrent parent N times. For each backcross generation, screen for the target traits, and select and retain the individuals with the target traits and the excellent characteristics of the recurrent parent for subculture to obtain the introgressed material BCN generation. Step 4: Self-cross the BCN generation obtained in Step 3 to obtain the B1 generation, and screen and retain the individuals with the target traits and the excellent characteristics of the recurrent parent. Step 5: Perform parthenogenesis and testcross on the B1 generation obtained in Step 4 at the same time. According to the testcross results, select and retain the B2 generation parthenogenesis line individuals with the target traits and the excellent characteristics of the recurrent parent for subculture to establish a pure parthenogenesis line.

2. The method for selecting and breeding silkworm varieties by using parthenogenesis assistance according to claim 1, characterized in that, The said Step 5 further includes: Step 51: If there is no B2 generation egg circle single moth with homozygous target traits in Step 5, repeat Step 4 and Step 5 until offspring with homozygous target traits and retaining the excellent characteristics of the recurrent parent appear.

3. A method for assisting in the breeding of silkworm varieties by parthenogenesis as described in claim 1 or 2, characterized in that Also including the steps: Step 61: Self-cross the B1 generation obtained in Step 4 to obtain the B2 generation self-cross population. Step 62: Screen the male individuals in the B2 generation self-cross population that retain the target traits and the excellent characteristics of the recurrent parent, and prepare the B3 generation and the testcross material in the way of mating one male with two females in a numbered manner. The female parent of the B3 generation bisexual reproduction line is the pure parthenogenesis line individual obtained in Step 5. Step 63: According to the testcross results, select and retain the B3 generation individuals with the target traits and the excellent characteristics of the recurrent parent for subculture to establish a pure bisexual reproduction line.

4. The method for assisting in the breeding of silkworm varieties by parthenogenesis as described in claim 3, characterized in that, Also including the steps: Step 64: If there is no B3 generation egg circle single moth with homozygous target traits in Step 62, repeat Step 4 and Steps 61 - 62 until offspring with homozygous target traits and retaining the excellent characteristics of the recurrent parent appear.

5. A method for assisting in the breeding of silkworm varieties by parthenogenesis as described in claim 1, characterized in that, The said target traits are detectable, easy to judge, heritable, and relatively stable traits. The traits include dominant traits controlled by single genes, polygenes without major genes, polygenes with major genes, as well as single traits controlled by single genes and multiple traits controlled by multiple genes.

6. The method for assisting in the breeding of silkworm varieties by parthenogenesis as described in claim 1, wherein, The said recurrent parent can be a non-parthenogenesis high-incidence material or a parthenogenesis high-incidence material.

7. The method for selecting and breeding silkworm varieties assisted by parthenogenesis according to claim 1, characterized in that, The said recurrent parent can be a single parent or multiple parents.

8. A method for breeding silkworm varieties assisted by parthenogenesis as described in claim 1, characterized in that, The number of backcrosses N≥3.

9. A method for breeding silkworm varieties assisted by parthenogenesis as described in claim 1, characterized in that, In the gene composition of the offspring after backcrossing N times, the gene composition of the parthenogenesis high-incidence material is ≤3.12%; if the recurrent parent is a parthenogenesis high-incidence material, the gene composition of the recurrent parent is ≥96.88%.

Images