Method for assisting breeding of silkworm varieties by using parthenogenesis

Abstract

The invention relates to the technical field of new silkworm variety breeding, and discloses a method for assisting silkworm variety breeding by parthenogenesis, which comprises the following steps: introducing a parthenogenesis high-incidence material into a target variety, screening individual hybridization recurrent parents retaining target characters or genes, and backcrossing with recurrent parent varieties, excellent individuals with target characters are selected for parthenogenesis homozygous immobilization and bisexual reproduction homozygous immobilization; according to the characteristic that a parthenogenesis high-incidence material is not easy to lose in filial generations due to the parthenogenesis high incidence, the parthenogenesis high-incidence material is hybridized, backcrossed and selfed with a target character variety and a conventional excellent variety, and after backcrossing is completed, parthenogenesis homozygous target character is carried out; target character homozygosis can be realized in a few generations, stable homozygous offspring can be obtained, and the breeding process is expected to be shortened.

Description

Technical Field

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

[0002] Silkworm breeding is the 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 science and technology, breeding technology has also made great progress. Molecular marker-assisted selection, gene editing technology and molecular design breeding technology are also being introduced into silkworm breeding. As an unconventional method, parthenogenesis has always attracted the attention of silkworm breeders because of its unique advantages. Parthenogenesis refers to the process in which female gametes directly induce single cell division and develop into embryos under the control of genes or external factors without fertilization. Therefore, parthenogenesis can quickly homozygous and fix genes and traits.

[0003] Scholars from various countries have conducted a lot of research on the artificial induction of parthenogenesis in silkworms. In particular, Astaurov explored the temperature and time parameters of heat stimulation to induce parthenogenesis in silkworms, effectively improving the incidence and hatching rate of parthenogenesis. Wang Yongqiang, Du Xin, Weng Hongbiao, Song Rongsu and others further optimized the induction method of parthenogenesis, invented the corresponding egg retrieval equipment, created and conserved a large number of materials with high incidence of parthenogenesis, and provided a strong guarantee for the role of parthenogenesis in the selection and breeding of silkworm varieties.

[0004] Patent CN202210785501.X discloses a method for breeding silkworm varieties resistant to blood-type pus disease, which uses disease-resistant genes to introduce into target varieties, select and retain disease-resistant genes in each generation, backcross with target varieties to retain economic traits of target varieties, and detect homozygous disease-resistant genes by mating one male with two females. Similar methods have a small adaptability and are only used for introducing disease-resistant genes into target varieties. Moreover, the maternal disease-resistant genes are not easy to be homozygous. If the maternal disease-resistant genes are not homozygous in the B2 generation, it is necessary to raise the egg circle single moths with homozygous paternal disease-resistant genes in the B3 generation, and perform male and female tests in a cycle until the maternal disease-resistant genes are detected to be homozygous. This method has the disadvantages of uncertainty, large workload, and long breeding time.

[0005] In the face of the above-mentioned technological gaps, there is an urgent need to further study the methods of parthenogenetic assisted variety selection and find a completely new method for silkworm variety selection to simplify experimental design and technical operations, accelerate the fixation of target genes, avoid the accumulation of adverse genes or the loss of beneficial genes, and shorten the breeding cycle. Summary of the invention

[0006] In order to solve the above-mentioned existing problems, the present invention discloses a method for utilizing parthenogenesis to assist in the breeding of silkworm varieties, which solves the problem that the current breeding methods may involve complex experimental designs and technical operations, resulting in high costs, long breeding cycles, and long-term excessive selection of materials or homozygosity may lead to the accumulation of certain unfavorable genes or the loss of beneficial genes.

[0007] The present invention provides a method for breeding silkworm varieties using parthenogenesis, wherein a parthenogenesis high-incidence material is introduced into a target variety, individuals retaining target traits or genes are screened for hybridization with recurrent parents, backcrossed with the recurrent parent variety, and excellent individuals with target traits are selected for parthenogenesis homozygous fixation and bisexual reproduction homozygous fixation. The specific steps are as follows:

[0008] Step 1: Use parthenogenetic materials with high occurrence rates to cross with target varieties and screen individuals that retain target traits;

[0009] Step 2: hybridizing the individuals retaining the target trait described in step 1 with the recurrent parent to obtain the F1 generation;

[0010] Step 3: Use the F1 generation described in step 2 to perform N backcrossings with the recurrent parent, screen the target traits for the offspring each time, select the individuals that retain the target traits and contain the excellent characteristics of the recurrent parent to obtain the BCN generation of the transformed material;

[0011] Step 4: self-pollinate the BCN generation described in step 3 to obtain the B1 generation, and screen individuals that retain the target trait and contain the excellent characteristics of the recurrent parent;

[0012] Step 5: Use the B1 generation described in step 4 to perform parthenogenesis and test cross simultaneously, and select B2 generation parthenogenetic individuals with target traits and excellent characteristics of the recurrent parent for succession based on the test cross results to establish a homozygous parthenogenetic line.

[0013] The high-incidence material of parthenogenesis is a silkworm variety with a high incidence of parthenogenesis, and the characteristics of high incidence of parthenogenesis are not easily lost in the offspring, which comes from the previous research of the inventor. 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 Zhejiang Feng No. 1, female 35 in Zhejiang Feng No. 2, and female 35CN in Zhejiang Feng 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 solution of the present invention.

[0014] The target variety is a silkworm variety containing target traits. Generally speaking, the target traits are detectable, easily determined, heritable, and relatively stable traits, such as disease resistance, fluoride resistance, markings, blood color, etc. The traits can be dominant traits controlled by a single gene, multiple genes without a major effect gene, or multiple genes with a major effect gene. It can be a single trait controlled by a single gene or multiple traits controlled by multiple genes. Traits that are obviously affected by environmental factors and epigenetics are not included. 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] In backcrossing (the hybrid produced by crossing two parents and then crossing one of the parents), generally a variety with excellent characteristics is selected as the female parent in the first cross, and as the male parent in subsequent backcrosses. This parent is called the recurrent parent in the backcross. In the present invention, in order to avoid overly complicated descriptions, all hybrid parents involved are referred to as recurrent parents.

[0016] Step 5 proposes that parthenogenesis and test cross be carried out simultaneously, making full use of the fact that there are still eggs remaining in the body of the female moth after laying eggs through temperature change stimulation, and conducting test crosses based on this, which can quickly screen whether the maternal target trait is homozygous or not; in most cases, a homozygous parthenogenetic system can be directly obtained, and the probability that all successive individuals are heterozygous is very low. A homozygous parthenogenetic system can also be obtained by repeating steps 4 and 5 once; overall, parthenogenesis assistance can homozygous the target gene to the B2 generation, while conventional methods require after the B3 generation, and the success rate of parthenogenesis assistance is significantly improved.

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

[0018] In some embodiments, step 5 further comprises:

[0019] Step 51: If there is no B2 generation egg-ringed moth homozygous for the target trait in step 5, repeat steps 4 and 5 until an offspring homozygous for the target trait and retaining the excellent characteristics of the recurrent parent appears.

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

[0021] In some embodiments, the method further comprises the steps of:

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

[0023] Step 62: Screening male individuals with target traits and excellent characteristics of the recurrent parent in the B2 generation selfing population, and preparing B3 generation and test cross materials in a way of mating one male with two females in pairs, wherein the female parent of the B3 generation bisexual reproductive system is the homozygous parthenogenetic reproductive system individual obtained in step 5;

[0024] Step 63: Based on the test cross results, select B3 generation individuals with target traits and excellent characteristics of the recurrent parent for succession to establish a homozygous bisexual reproductive line.

[0025] By adopting the technical scheme of the present invention, parthenogenesis and female individual test cross are performed in the B1 generation where homozygous individuals appear, and homozygous female individuals are quickly obtained. If a bisexual reproductive system needs to be established, it is only necessary to test cross the male individuals in the B2 generation, without complicated experimental design and technical operation, and the accumulation of undesirable genes in the breeding materials can be avoided to a certain extent, ensuring that excellent traits are not lost, reducing scientific research costs, and shortening the breeding process.

[0026] In some embodiments, the method further comprises the steps of:

[0027] Step 64: If there is no B3 generation egg-ringed moth homozygous for the target trait in step 62, repeat steps 4 and steps 61-62 until an offspring homozygous for the target trait and retaining the excellent characteristics of the recurrent parent appears.

[0028] Similarly, the reason for repeating step 64 is that the successive individuals may all be heterozygous, although this possibility is very small; a homozygous hermaphroditic reproductive system can be obtained by repeating hermaphroditic reproduction and test cross once.

[0029] One of the core applications of the present invention is to conveniently introduce multiple different target traits into new varieties of silkworms: a B1 generation parthenogenetic reproductive system containing multiple target trait genes is obtained by hybridization, backcrossing and selfing of multiple target varieties, and the homozygosity of multiple target trait genes in the B1 generation parthenogenetic reproductive system can be verified by a single test cross; when the constructed parthenogenetic reproductive system is verified to be homozygous for multiple target trait genes through a test cross, it can be sexually reproduced with males of a B2 generation selfing group, and simultaneously test crossed with males of the sexual reproductive group, and a sexual reproductive system homozygous for multiple target traits can be constructed based on the test cross results.

[0030] When it comes to the aggregation breeding of multiple target traits, conventional breeding methods require multiple test crosses in each generation, each time verifying the homozygous gene of the target trait of the father or mother, so the number of test crosses increases exponentially with the increase in the number of target traits, which is one of the difficulties of current aggregation breeding; however, when the method of the present invention is adopted, due to the stable genetic properties of the parthenogenetic reproductive system, only two test crosses are required to obtain a homozygous bisexual reproductive system. Compared with conventional breeding methods, the method of the present invention has significant advantages in aggregation breeding and significantly shortens the breeding cycle.

[0031] In some embodiments, the parthenogenesis high incidence material has a parthenogenesis incidence of ≥90%, a hatching rate of ≥85%, and stable inheritance, and is relatively dominant to the low incidence.

[0032] The meaning of "relative dominance to low incidence" is that the incidence of parthenogenesis is a quantitative trait, and the dominant and recessive relationship between high and low incidence is not absolute, but may show dynamic changes due to different genetic interactions, 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 methods, the target trait is a detectable, easily determined, heritable, and relatively stable trait, and the trait includes a dominant trait controlled by a single gene, multiple genes without a major effect gene, or multiple genes with a major effect gene. The trait includes a single trait 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-parthenogenesis high-incidence material) or a parthenogenesis high-incidence material.

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

[0037] In some embodiments, the number of backcrossings N is ≥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. Taking advantage of the fact that the high incidence of parthenogenesis is not easily lost in the hybrid offspring, the high incidence of parthenogenesis is hybridized, backcrossed and selfed with the target trait varieties and conventional excellent varieties, and then the homozygous target trait of parthenogenesis is carried out after the backcrossing is completed. The homozygous target trait can be achieved in fewer generations, and stable homozygous offspring can be obtained, which is expected to shorten the breeding process;

[0045] 2. The female moth still has eggs left in its body after being stimulated by temperature changes to lay eggs. Based on this, synchronous parthenogenesis and test cross can be performed to quickly screen whether the target traits of the mother are homozygous or not; in the most likely case, a homozygous parthenogenetic system can be directly obtained. In the very low probability case, all the successive individuals are heterozygous, and a homozygous parthenogenetic system can also be obtained by repeating parthenogenesis and test cross once; in general, parthenogenesis assisted to the B2 generation can homozygous target genes, while conventional methods require after the B3 generation, and the success rate of parthenogenesis assisted is significantly improved;

[0046] 3. If it is necessary to establish a bisexual reproductive system, it is only necessary to test cross the male individuals in the B2 generation, without complicated experimental design and technical operation. This can avoid the accumulation of undesirable genes in the 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 polymer breeding of multiple target traits, conventional breeding methods require multiple test crosses in each generation, each time verifying the homozygosity of the paternal or maternal target trait gene of a certain target trait. Therefore, the number of test crosses increases exponentially with the increase in the number of target traits, which is one of the difficulties of current polymer breeding. However, when the method of the present invention is adopted, due to the stable genetic characteristics of the parthenogenetic system, only two test crosses are required to obtain a homozygous hermaphroditic reproductive system: first, the B1 generation parthenogenetic system is subjected to parthenogenesis and test crosses at the same time, and a homozygous parthenogenetic system can be constructed based on the test cross results. The homozygous parthenogenetic system is subjected to hermaphroditic reproduction with B2 generation male individuals, and test crosses are performed on B2 generation male individuals at the same time, and a homozygous hermaphroditic reproductive system can be constructed based on the test cross results. Compared with conventional breeding methods, the method of the present invention has significant advantages in polymer breeding and significantly shortens the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Flow chart of the method for utilizing parthenogenesis to assist silkworm variety selection of the present invention DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific examples and accompanying drawings, which 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 examples are reagents and materials available from commercial sources 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 Individuals / ML of silkworm blood-type pus disease polyhedron suspension were fed with mulberry leaves for 24 hours, and excellent individuals were selected 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 Individuals / ML of silkworm blood-type pus disease polyhedron suspension were fed with mulberry leaves for 24 hours, and excellent individuals were selected 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 with the suspension of polyhedral body of blood-type pus disease of silkworm were added for 24 hours, and the homozygous disease resistance gene of the parthenogenetic offspring was tested. After testing, all the individuals in 13 egg circles (moth areas) of the test cross offspring survived, indicating that the disease resistance gene of the female moths corresponding to the numbers of the 13 egg circles was homozygous, and the corresponding female moths were selected to construct the parthenogenetic system with homozygous blood-type pus disease resistance gene.

[0058] The construction of a homozygous parthenogenetic line of resistance to blood-type pyogenic disease is also a kind of aggregate breeding, specifically, the high parthenogenetic incidence trait of female 35 and the disease resistance trait of Qiufeng N are simultaneously introduced into new silkworm varieties. Based on the same principle, more different target traits can also be introduced into new silkworm varieties, especially when the number of target traits is large, and the B1 generation parthenogenetic line containing multiple target trait genes can be obtained by hybridization, backcrossing and self-pollination of multi-target trait varieties and high-incidence parthenogenetic materials. The homozygosity of multiple target trait genes in the B1 generation parthenogenetic line can be verified by a single test cross, and the corresponding B2 generation parthenogenetic individuals can be screened to construct a parthenogenetic line homozygous for multiple target trait genes.

[0059] Example 2

[0060] Take the B1 generation in Example 1, add poison to feed, select excellent individuals for self-pollination, and obtain the B2 generation self-pollination group. Select 25 male moths with excellent individuals, and mate one male with two females, one of which is the B2 generation parthenogenetic offspring in Example 1, and the other female is the female moth of the test cross material. In a high temperature and high humidity environment, the B2 generation self-pollination group and the test cross material offspring ant silkworms were coated with 1.5×10 7 The mulberry leaves with 100 / ML Bombyx mori blood-type pus disease polyhedron suspension were fed for 24 hours, and the homozygous disease resistance gene of the sexual offspring was tested. After testing, all the individuals in 10 egg circles (moth areas) of the test cross progeny survived, indicating that the disease resistance genes of the male moths corresponding to the numbers in the 10 egg circles were homozygous, and the corresponding B3 generation sexual offspring were selected to construct the homozygous sexual reproductive system of the blood-type pus disease resistance gene.

[0061] Based on the same principle, a bisexual reproductive system homozygous for multiple target trait genes can be constructed. When the constructed parthenogenetic reproductive system is verified to be homozygous for multiple target trait genes through test cross, it can be used for bisexual reproduction and synchronous test cross with males in the B2 generation selfing population in the form of one male mating with two females. Based on the test cross results, a bisexual reproductive system homozygous for multiple target traits can be constructed.

[0062] Comparative Example 1

[0063] The target variety and breeding method are the same as those in Example 1, except that the parthenogenetic high-incidence material of the present invention is not used, but conventional materials (non-parthenogenetic high-incidence materials) are used. In the spring of 2017, 40 female moths and 40 male moths with excellent individuals are selected from the B1 generation, and these 40 male moths are marked one by one, numbered 1 to 40, and the self-fertilized seed B2 generation and test cross materials are prepared in the form of one male and two females mating in pairs, and the eggs laid by the female moths are refrigerated and stored; the self-fertilized male moths are then tested with the female moths of the virus-sensitive variety Kasuga B, and the self-fertilized seed and the test cross male moths correspond one to one. In the summer of 2017, the test cross material was tested for homozygous disease resistance genes of the paternal parent. After testing, all individuals in 5 egg circles (moth areas) survived. The corresponding self-pollinated B2 generation egg circles have 5 egg circles that are homozygous for the paternal disease resistance gene; in the mid-autumn festival of 2017, the single moths in the egg circles that are homozygous for the paternal disease resistance gene were fed with poison until they emerged, and 40 male and female moths with excellent individuals were selected from each moth area, and the male and female moths were numbered respectively. The self-pollinated seed B3 generation and test cross materials were prepared in the way of mating one male with two females in pairs. Specifically, the male moths were separated after self-pollination with the female moths in the same moth area, that is, the same egg circle, and the self-pollinated B3 generation was marked and refrigerated for storage; the self-pollinated male moths were then test-crossed with the female moths of the virus-sensitive variety, and the egg circles laid by the female moths after the test cross were numbered one by one. In the late autumn of 2017, the test cross materials were tested for homozygosity of the maternal and paternal disease resistance genes, that is, the male and female were tested together. If all individuals in all egg circles (moth zones) survive, the corresponding B2 generation moth zones are homozygous for the maternal disease resistance gene (the paternal disease resistance gene was already homozygous in the previous round of testing); if all individuals in a certain egg circle (moth zone) survive, then the corresponding B3 generation egg circle is homozygous for the paternal disease resistance gene.

[0064] Compared with Comparative Example 1, the advantages of Example 1-2 are as follows: ⑴ The process of variety breeding is shortened, and parthenogenesis assistance can reach homozygous target genes in the B2 generation, while the method adopted in Comparative Example 1 requires after the B3 generation; ⑵ The success rate of parthenogenesis assistance is significantly improved; ⑶ Fully utilizes the residual eggs in the body of the female moth after laying eggs through temperature change stimulation, and based on this, synchronous parthenogenesis and test cross are carried out, which can quickly detect whether the maternal target trait is homozygous or not; ⑷ It is not limited to the parthenogenetic system, and a bisexual reproductive system can also be established as needed. It only needs to test cross the male individuals in the B2 generation, without complicated experimental design and technical operations, which can avoid the accumulation of non-desirable 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 aggregate breeding of multiple target traits, conventional breeding methods require multiple test crosses in each generation, and each time the homozygous verification of the target trait gene of the paternal or maternal parent of a certain target trait is performed, so the number of test crosses increases exponentially with the increase in the number of target traits, which is one of the difficulties of current aggregate breeding; however, when the method of the present invention is adopted, due to the stable genetic properties of the parthenogenetic system, only two test crosses are required to obtain a homozygous hermaphroditic reproductive system: first, the B1 generation parthenogenetic system is subjected to parthenogenetic reproduction and test crosses at the same time, and a homozygous hermaphroditic reproductive system can be constructed according to the test cross results, and the homozygous parthenogenetic system is subjected to hermaphroditic reproduction with the B2 generation male individuals, and the B2 generation male individuals are tested at the same time, and a homozygous hermaphroditic reproductive system can be constructed according to the test cross results. Compared with conventional breeding methods, the method of the present invention has significant advantages in aggregate breeding and significantly shortens the breeding cycle.

[0066] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

Claims

1. A method for selecting silkworm varieties using parthenogenesis, characterized in that: Includes steps: Step 1: Use parthenogenetic materials with high occurrence rates to cross with target varieties and screen individuals that retain target traits; Step 2: hybridizing the individuals retaining the target trait described in step 1 with the recurrent parent to obtain the F1 generation; Step 3: Use the F1 generation described in step 2 to perform N backcrossings with the recurrent parent, screen the target traits for the offspring each time, select the individuals that retain the target traits and contain the excellent characteristics of the recurrent parent to obtain the BCN generation of the transformed material; Step 4: self-pollinate the BCN generation described in step 3 to obtain the B1 generation, and screen individuals that retain the target trait and contain the excellent characteristics of the recurrent parent; Step 5: Use the B1 generation described in step 4 to perform parthenogenesis and test cross simultaneously, and select B2 generation parthenogenetic individuals with target traits and excellent characteristics of the recurrent parent for succession based on the test cross results to establish a homozygous parthenogenetic line.

2. The method for selecting silkworm varieties by using parthenogenesis as claimed in claim 1, characterized in that: The step 5 also includes: Step 51: If there is no B2 generation egg-ringed moth homozygous for the target trait in step 5, repeat steps 4 and 5 until an offspring homozygous for the target trait and retaining the excellent characteristics of the recurrent parent appears.

3. A method for selecting silkworm varieties using parthenogenesis as claimed in claim 1 or 2, characterized in that: Also includes the steps: Step 61: self-pollinating the B1 generation described in step 4 to obtain a B2 generation self-pollinating population; Step 62: Screening male individuals with target traits and excellent characteristics of the recurrent parent in the B2 generation selfing population, and preparing B3 generation and test cross materials in a way of mating one male with two females in pairs, wherein the female parent of the B3 generation bisexual reproductive system is the homozygous parthenogenetic reproductive system individual obtained in step 5; Step 63: Based on the test cross results, select B3 generation individuals with target traits and excellent characteristics of the recurrent parent for succession to establish a homozygous bisexual reproductive line.

4. The method for selecting silkworm varieties by using parthenogenesis as claimed in claim 3, characterized in that: Also includes the steps: Step 64: If there is no B3 generation egg-ringed moth homozygous for the target trait in step 62, repeat steps 4 and steps 61-62. Until offspring appear that are homozygous for the target trait and retain the excellent characteristics of the recurrent parent.

5. A method for selecting silkworm varieties using parthenogenesis as claimed in any one of claims 1 to 4, characterized in that: The parthenogenesis high incidence material has a parthenogenesis incidence of ≥90%, a hatching rate of ≥85%, and is stably inherited and relatively dominant to low incidence.

6. A method for selecting silkworm varieties using parthenogenesis as claimed in any one of claims 1 to 4, characterized in that: The target trait is a detectable, easily determined, heritable, and relatively stable trait, and the trait includes dominant traits controlled by single genes, multiple genes without major effect genes, and multiple genes with major effect genes, as well as single traits controlled by a single gene and multiple traits controlled by multiple genes.

7. A method for selecting silkworm varieties using parthenogenesis as claimed in any one of claims 1 to 4, characterized in that: The recurrent parent may be a non-parthenogenetic high-incidence material or a parthenogenetic high-incidence material.

8. A method for selecting silkworm varieties using parthenogenesis as described in any one of claims 1 to 4, characterized in that: The recurrent parent may be a single parent or multiple parents.

9. A method for selecting silkworm varieties using parthenogenesis as claimed in any one of claims 1 to 4, characterized in that: The backcrossing number N is ≥3.

10. A method for selecting silkworm varieties using parthenogenesis as claimed in any one of claims 1 to 4, characterized in that: In the gene composition of the offspring after N backcrossings, the gene composition of the material with a high incidence of parthenogenesis is ≤3.12%; if the recurrent parent is a material with a high incidence of parthenogenesis, the gene composition of the recurrent parent is ≥96.88%.

Images