A method for grading and identifying silkworm germplasm resources

Abstract

The application discloses a method for grading and identifying silkworm germplasm resources. According to genetic differences in molecular evolution among silkworm germplasm resources, a grading and identifying method is established by using preferred specific SSR molecular markers, which can first distinguish different geographical populations, then distinguish germplasm resource groups in the same geographical population, and finally distinguish all silkworm germplasm resources in the same germplasm resource group. The method overcomes the problem that the prior art method cannot simultaneously establish specific DNA molecular markers for a large number of germplasm resources, and cannot accurately identify and distinguish specific germplasm resources.

Description

Technical Field

[0001] This invention relates to a method for grading and identifying silkworm germplasm resources, belonging to the field of sericulture. Background Technology

[0002] One effective measure to protect plant and animal varieties is to construct DNA fingerprint profiles and specific DNA molecular markers for each variety. Constructing DNA fingerprint profiles and specific DNA molecular markers for silkworm varieties is an effective means of protecting the intellectual property rights of breeders and owners of new silkworm varieties, and also serves as an objective standard for monitoring the safety of silkworm variety production and use. Establishing DNA molecular markers and identification technologies for silkworm varieties can accelerate the breeding of superior silkworm varieties, avoid breeding errors such as inbreeding, and, more importantly, effectively protect the interests of those involved in the variety, thus accelerating the promotion and use of silkworm varieties.

[0003] However, establishing a DNA fingerprint and specific DNA molecular markers for a new silkworm variety currently lacks the support of a relevant public database of silkworm varieties. On the other hand, the number of silkworm varieties is enormous and the types are diverse, making it difficult to establish DNA fingerprints and variety-specific DNA molecular markers covering the vast majority of silkworm varieties in the short term.

[0004] Silkworms have a history of breeding and selection spanning thousands of years. During this long process of adaptation and differentiation, different geographical populations have emerged due to geographical isolation in their rearing areas, including Chinese, Japanese, tropical, and European varieties. Among these, the Chinese and Japanese varieties are the most numerous and widely used in my country. Within each geographical population, due to natural evolution and human selection, genetic resources and production varieties with significant differences in biological and economic traits have emerged; these are collectively referred to as germplasm resources (varieties).

[0005] Silkworm germplasm resources are the source of new variety breeding. Establishing DNA fingerprinting or variety-specific DNA molecular markers for important silkworm germplasm resources is beneficial for studying the phylogenetic relationships and molecular phylogeny among germplasm resources. More importantly, it enables the identification, protection, and utilization of silkworm germplasm resources, and has significant practical value for the construction of core germplasm resources and the breeding of new silkworm varieties. Therefore, establishing reliable DNA molecular markers for silkworm germplasm resources is not only an effective measure for the protection of core silkworm germplasm resources, but also provides clues for breeding new varieties using silkworm germplasm resources.

[0006] The genetic differences among different germplasm resources of the same organism exist at multiple levels, including DNA coding and replication regulation, RNA transcription regulation and sequence differences, and protein translation and structural information. However, the theory of molecular genetics generally believes that the differences in DNA sequence information among different germplasm resources of the same organism are ubiquitous and can be stably detected.

[0007] Among the currently available publicly available technologies, the DNA molecular marker methods that can be used for different germplasm resources (varieties) of silkworm include: restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), single nucleotide polymorphism (SNP), and simple sequence repeats (SSR).

[0008] Including SSRs, also known as short tandem repeats (STRs) or microsatellites, existing DNA molecular marker technologies can obtain DNA molecular markers for different germplasm resources (varieties) of most agricultural organisms, including silkworms, and can conduct genotyping and pedigree analysis of dozens or even more germplasm resources, especially the study of evolutionary relationships of germplasm resources (varieties). However, the identification and differentiation of germplasm resources (varieties) can only be carried out within a very small number of germplasm resources (varieties). Within a large number of germplasm resources (varieties), such as more than 20, there is no publicly available method that can simultaneously establish specific DNA molecular markers for all germplasm resources (varieties) and use them to accurately distinguish and identify specific germplasm resources (varieties). Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a grading and identification method that utilizes selected silkworm germplasm resources (varieties)-specific SSR molecular markers. This method first distinguishes different geographical populations, then further differentiates germplasm resource (variety) groups within the same geographical population, and finally identifies and distinguishes all germplasm resources (varieties) within a germplasm resource (variety) group. This invention's technical method is convenient to use, low in cost, and provides accurate identification.

[0010] The first objective of this invention is to provide a method for grading and identifying silkworm germplasm resources, comprising the following steps:

[0011] S1. Amplify the silkworm DNA to be identified using primers as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4, and classify it once based on the polymorphism of the amplification product;

[0012] S2. Based on the results of the first classification in S1, a second classification is performed using primers as shown in SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, or SEQ ID NO.11-12.

[0013] The inventive principle of this invention is as follows:

[0014] Although the number of silkworm germplasm resources (varieties) is vast, existing DNA molecular marker methods such as SSR (Sequencing Research Signs) are insufficient for simultaneous DNA molecular marker identification of all germplasm resources (varieties). Existing silkworm germplasm resources (varieties) belong to four distinct geographical populations: Chinese, Japanese, tropical, and European. Within each geographical population, multiple germplasm resources (varieties) exhibit both molecular evolutionary aggregation and dispersion biases, allowing for further subdivision into different germplasm resource (variety) groups. By selecting specific SSR molecular markers for silkworm germplasm resources (varieties), it is possible to first differentiate the numerous silkworm germplasm resources (varieties) by geographical population. Further, it is possible to differentiate multiple germplasm resources (varieties) within the same geographical population into a limited number (less than 20) of germplasm resource (variety) groups. Then, under current technological conditions, it is possible to establish unique molecular tags (fingerprints) for each silkworm germplasm resource (variety) within a germplasm resource (variety) group. Based on these unique molecular tags (fingerprints), germplasm resource (variety) identification and intellectual property protection can be achieved.

[0015] This invention innovatively classifies and identifies silkworms based on the stable genetic characteristics resulting from their unique geographical regions. First, high-throughput screening yields SSR primers (sequences 1-4), enabling efficient identification (primary classification) of Chinese and Japanese geographical species. Specifically, sequences 1-2 and 3-4 can achieve a "1 or 0" identification. When using sequences 1-2 to amplify the test DNA, if it is a Chinese geographical species, an amplification product will be observed; otherwise, no SSR sequence product will be present. The same applies when using sequences 3-4 to amplify Japanese geographical species. The polymorphism of the products makes it very easy to distinguish silkworms from different geographical species. Second, during secondary classification, the amplified bands (length, arrangement, etc.) are further subdivided to identify different germplasm resources (varieties) within the same geographical population.

[0016] Further, optionally, based on the secondary classification, a tertiary classification can be performed using primers as shown in SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, or SEQ ID NO.23-24. Tertiary classification can distinguish different genomic resources within the same group of genomic resources (varieties).

[0017] Furthermore, a single classification was used to identify geographical species from China, Japan, tropical regions, and Europe.

[0018] Furthermore, the PCR products were displayed by electrophoresis, and the tested silkworms were distinguished by comparison with standard electrophoresis patterns.

[0019] Furthermore, the electrophoretic products were visualized using silver staining.

[0020] Furthermore, the samples from which the silkworm DNA to be tested was extracted were selected from eggs, larvae, pupae, or adults.

[0021] Furthermore, during classification, one or more sets of primers are selected for identification.

[0022] Furthermore, the amplification reaction system includes a DNA template, amplification primers, Taq DNA polymerase, and dNTPs.

[0023] The second objective of this invention is to provide an SSR molecular library for identifying silkworm germplasm resources, wherein the SSR molecular library includes at least the primer sequences shown in SEQ ID NO.1-12.

[0024] Furthermore, the SSR molecular library also includes the primer sequences shown in SEQ ID NO.13-24.

[0025] The beneficial effects of this invention are:

[0026] (1) Using the method of this invention, specific germplasm resources (varieties) can be quickly and accurately identified from a large number of silkworm germplasm resources (varieties), and different strains of the same silkworm resource (variety) can also be identified. It can avoid breeding errors such as inbreeding and accelerate the breeding speed of superior silkworm varieties.

[0027] (2) Using the method of this invention, DNA molecular variations of the same silkworm resource (variety) in different preservation environments (bases) can be detected. It can monitor molecular genetic drift that occurs during the preservation and use of silkworm germplasm resources (variety) and correct deviations in the preservation of silkworm germplasm resources (variety).

[0028] (3) Using the method of the present invention, it is possible to supervise the loss of silkworm germplasm resources (variety) during the preservation process and correct the moth area mixing that occurs during the preservation process of silkworm germplasm resources (variety). Attached Figure Description

[0029] Figure 1The effect of the primary SSR primer sequences SEQ ID NO.1 / SEQ ID NO.2 (AS0940) on the identification of geographical populations of silkworm germplasm resources (varieties). In the figure, M represents the standard molecular weight marker. The 24 geographical species in China are numbered as follows: 1-Su5; 3-Su3; 4-Qiu3; 6-75Xin; 8-2A; 9-4A; 12-57A; 13-57B; 16-Huafeng; 18-Suju; 19-829Su; 20-827Ju; 23-ChunleiSu; 25-HuanghaiA; 26-HuanghaiB; 27-SuzhenA; 30-C497; 32-Huhua; 34-Wuhua; 36-C; 39-Su7; 40-Su9; 43-Su1; 44-Dong34Su. The 21 geographical species of Japan are numbered as follows: 2-Su6; 5-Su4; 7-7532Su; 10-1A; 11-3A; 14-24; 15-46; 17-Cedar; 21-7910A; 22-8214Tiger; 24-Shin-Fang A; 28-Haru-Kuang C; 29-Haru-Kuang D; 31-322; 33-Aki-Hoshi; 35-Hoshi-Hoshi; 37-F; 38-H; 41-Su8; 42-Su10; 45-Su12Su. The boxes in the figure indicate the characteristic polymorphic identification regions.

[0030] Figure 2 The figure shows the identification effect of the primary SSR primer sequences SEQ ID NO.3 / SEQ ID NO.4 (AS1102) on the geographical populations of silkworm germplasm resources (varieties). In the figure, M represents the standard molecular weight marker. Numbers 1-45 represent the silkworm germplasm resource (variety) numbers. Figure 1 Same. The area within the box in the figure represents the characteristic polymorphism recognition region.

[0031] Figure 3 The effect of secondary SSR primers on population differentiation of the Chinese geographical species of *Bombyx mori* is shown. (A) Primer sequence SEQ ID NO.5 / SEQ ID NO.6 (BS1603). (B) Primer sequence SEQ ID NO.7 / SEQ ID NO.8 (BS0905). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the *Bombyx mori* germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1 Same. The boxes and letters A, B, and C in the diagram represent different germplasm resource (variety) groups identified according to polymorphism in the geographical species of China.

[0032] Figure 4 The effect of secondary SSR primers on population differentiation of Japanese geographical species of silkworm is shown. (A) Primer sequence SEQ ID NO.9 / SEQ ID NO.10 (BS1504). (B) Primer sequence SEQ ID NO.11 / SEQ ID NO.12 (BS0305). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the silkworm germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1Same. The boxes and letters A, B, and C in the diagram represent different germplasm resource (varietal) groups identified according to polymorphism in the geographical classification of Japanese species.

[0033] Figure 5 The effectiveness of tertiary SSR primers in identifying seven silkworm germplasm resources (varieties) within the BS1603-A group of the Chinese geographical species of *Bombyx mori* was studied. (A) Primer sequence SEQ ID NO.13 / SEQ ID NO.14 (CS0203). (B) Primer sequence SEQ ID NO.15 / SEQ ID NO.16 (CS0601). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the *Bombyx mori* germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1 Same. The area within the box in the figure represents the characteristic polymorphism recognition region.

[0034] Figure 6 The effectiveness of tertiary SSR primers in identifying nine silkworm germplasm resources (varieties) within the BS1603-B group of the Chinese geographical species of *Bombyx mori* was studied. (A) Primer sequence SEQ ID NO.13 / SEQ ID NO.14 (CS0203). (B) Primer sequence SEQ ID NO.15 / SEQ ID NO.16 (CS0601). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the *Bombyx mori* germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1 Same. The area within the box in the figure represents the characteristic polymorphism recognition region.

[0035] Figure 7 The effectiveness of tertiary SSR primers in identifying eight silkworm germplasm resources (varieties) within the BS1603-C group of the Chinese geographical species of *Bombyx mori* was studied. (A) Primer sequence SEQ ID NO.13 / SEQ ID NO.14 (CS0203). (B) Primer sequence SEQ ID NO.15 / SEQ ID NO.16 (CS0601). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the *Bombyx mori* germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1 Same. The area within the box in the figure represents the characteristic polymorphism recognition region.

[0036] Figure 8 The effectiveness of tertiary SSR primers in identifying six silkworm germplasm resources (varieties) within the BS1504-A group of the Japanese geographical species of *Bombyx mori* was studied. (A) Primer sequence SEQ ID NO.15 / SEQ ID NO.16 (CS0601). (B) Primer sequence SEQ ID NO.17 / SEQ ID NO.18 (CS2708). (C) Primer sequence SEQ ID NO.19 / SEQ ID NO.20 (CS0901). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the *Bombyx mori* germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1 Same. The area within the box in the figure represents the characteristic polymorphism recognition region.

[0037] Figure 9 The effectiveness of tertiary SSR primers in identifying six silkworm germplasm resources (varieties) within the BS1504-B group of the Japanese geographical species of *Bombyx mori* was studied. (A) Primer sequence SEQ ID NO.15 / SEQ ID NO.16 (CS0601). (B) Primer sequence SEQ ID NO.17 / SEQ ID NO.18 (CS2708). (C) Primer sequence SEQ ID NO.19 / SEQ ID NO.20 (CS0901). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the *Bombyx mori* germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1 Same. The area within the box in the figure represents the characteristic polymorphism recognition region.

[0038] Figure 10 The effectiveness of tertiary SSR primers in identifying nine silkworm germplasm resources (varieties) within the BS1504-C group of the Japanese geographical species of *Bombyx mori* was studied. (A) Primer sequence SEQ ID NO.17 / SEQ ID NO.18 (CS2708). (B) Primer sequence SEQ ID NO.19 / SEQ ID NO.20 (CS0901). (C) Primer sequence SEQ ID NO.21 / SEQ ID NO.22 (CS1502). In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the *Bombyx mori* germplasm resource (variety) numbers, and the content of the numbers is consistent with... Figure 1 Same. The area within the box in the figure represents the characteristic polymorphism recognition region.

[0039] Figure 11 This study demonstrates the effectiveness of tertiary SSR primer pairs in identifying genetic variations among strains of the same silkworm germplasm resource (variety) during preservation. The tertiary SSR primer pairs are SEQ ID NO.15 / SEQ ID NO.16 (CS0601) and SEQ ID NO.23 / SEQ ID NO.24 (CS0201). Dark gray boxes indicate stable and consistent bands or regions among different strains or preservation units of the same variety; light gray boxes indicate bands or regions showing genetic variation among different strains or preservation units of the same variety. In the figure, M represents the standard molecular weight marker. Sample laboratory numbers include: germplasm resource (variety) number and strain identification code—preservation location identification code. 1, Su5 basic strain; 1Z, Su5Z strain; 1X, Su5X strain. HA and SH represent two germplasm resource (variety) preservation units from different regions.

[0040] Figure 12The identification effect of SSR primers on different silkworm germplasm resources (varieties) is shown. (A) Primer CS0101, (B) Primer CS0201, (C) Primer CS0302. In the figure, M is the standard molecular weight marker. Sample laboratory number content: germplasm resource (variety) number and strain identification code - preservation location identification code. 1-ZJ, Su5ZJ strain; 2-ZJ, Su6ZJ strain; 6-DT, 75 New DT strain; 7-DT, Su7532DT strain.

[0041] Figure 13 The effect of SSR primers on population differentiation of Chinese geographical species of silkworm. (A) Primer CS1103,

[0042] (B) Primer CS0607. In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the silkworm germplasm resource (variety) numbers, and the content of the numbers is the same as... Figure 1 Same. The boxes and letters A, B, and C in the diagram represent different germplasm resource (variety) groups identified according to polymorphism in the geographical species of China.

[0043] Figure 14 The effect of SSR primers on population differentiation of Chinese geographical species of silkworm. (A) Primer CS0505.

[0044] (B) Primer CS0504. In the figure, M represents the standard molecular weight marker. The numbers at the top of the figure are the silkworm germplasm resource (variety) numbers, and the content of the numbers is the same as... Figure 1 Same. The boxes and letters A, B, and C in the diagram represent different germplasm resource (variety) groups identified according to polymorphism in the geographical species of China. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0046] The primers used in the following examples are as follows:

[0047] Table 1. Specific SSR primers that can be used for graded identification of silkworm germplasm resources (varieties).

[0048]

[0049]

[0050] The solutions involved in this invention are as follows:

[0051] One objective of this invention is to provide a method for classifying and identifying a large number of silkworm germplasm resources (varieties) using SSR molecular identification markers. Another objective of this invention is to establish SSR molecular identification markers for all silkworm germplasm resources (varieties).

[0052] To achieve the above objectives, the present invention provides the following technical solution:

[0053] In one aspect, this invention provides a polymerase chain reaction (PCR) SSR primer screening method for DNA molecular marker identification of silkworm germplasm resources (varieties) based on SSR molecular marker technology, comprising the following steps:

[0054] (1) After a large number of DNA molecular marker polymorphism detection experiments, the SSR primers listed in Table 1 were selected from the silkworm SSR database for graded identification of candidate PCR amplification.

[0055] Each of the selected SSR primers can generate abundant DNA molecular marker polymorphisms in a large class of silkworm germplasm resources (varieties). The selected SSR primers are evenly distributed in 28 linkage groups of silkworm. The selected SSR primers can obtain the genetic differences in molecular evolution among silkworm germplasm resources (varieties) over a large range.

[0056] (2) Using the SSR primers listed in Table 1, DNA molecular marker polymorphism classification was performed on a large number of silkworm germplasm resources (variety), and specific SSR primers that can be used for graded identification of silkworm germplasm resources (variety) were selected (Table 1).

[0057] The selected specific SSR primers for classifying and identifying high-quality silkworm germplasm resources (varieties) are as follows: the first-level primers in Table 1 are suitable for identifying and distinguishing geographical populations of silkworm germplasm resources (varieties); the second-level primers are suitable for identifying and distinguishing different germplasm resource (variety) groups within the same geographical population; and the third-level primers are suitable for identifying and distinguishing different silkworm germplasm resources (varieties) within a single germplasm resource (variety) group.

[0058] On the other hand, the present invention provides a method for DNA molecular markers and identification of silkworm germplasm resources (varieties), comprising the following steps:

[0059] (1) Extract the whole genome DNA of the silkworm germplasm resource (variety) to be identified to obtain the DNA template, specifically including:

[0060] (a) Processing of biological samples of eggs, larvae, pupae or adults of silkworm germplasm resources (varieties).

[0061] Preferably, egg samples of the silkworm germplasm resources (varieties) to be identified are incubated at 25℃ (±3℃) and 65-85% relative humidity until the embryos inside the eggs develop to the greening stage. Dead and unfertilized eggs are then removed, and the greening eggs are collected for disinfection. Preferably, the egg surface is disinfected by soaking in 2-4% (v / v) glutaraldehyde for 15 minutes, followed by rapid washing twice with 100% anhydrous ethanol. After drying on a sterile operating table, the eggs are stored at -80℃ for later use.

[0062] Preferably, after disinfecting the body surface of the larval, pupa, or adult biological samples of the silkworm germplasm resources (variety) to be identified, the epidermis is broken open with a scalpel in a sterile operating table, and the silk gland, fat body, or gonadal tissue is extracted with surgical forceps. The extracted tissue is washed twice with sterilized physiological saline, and the surface moisture is absorbed with absorbent paper before storage at -80°C. Preferably, the body surface of the larval, pupa, and adult biological samples is disinfected by soaking in 75% ethanol for 5 minutes and then washing twice with sterilized physiological saline. Preferably, before soaking in 75% ethanol, the head and tail of the larvae, pupae, and adults are tied tightly with fine thread to prevent internal waste from leaking out of the mouthparts or anus during the disinfection process.

[0063] (b) Extract the whole genome DNA of silkworm germplasm resources (varieties) to obtain DNA templates.

[0064] Preferably, an excess of egg samples, or larval, pupa, or adult tissue samples, are placed in a sterilized mortar pre-cooled with liquid nitrogen, and ground into powder with liquid nitrogen. 1000 mg of the powder is weighed and transferred to a sterile centrifuge tube. Using a commercially available DNA extraction kit, the whole-genome DNA of the silkworm is extracted according to the kit's instructions, while simultaneously removing RNA. The extracted DNA sample is stored at -20°C.

[0065] (2) PCR reaction to amplify the SSR sequence in the whole genome DNA of the silkworm germplasm resource (variety) to be identified. The primers used in the PCR reaction include at least one or more of the following sequence DNA primers.

[0066] Primary SSR primers: upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2

[0067] Upstream primer SEQ ID NO.3 and downstream primer SEQ ID NO.4; secondary SSR primers: upstream primer SEQ ID NO.5 and downstream primer SEQ ID NO.6.

[0068] Upstream primer SEQ ID NO.7 and downstream primer SEQ ID NO.8

[0069] Upstream primer SEQ ID NO.9 and downstream primer SEQ ID NO.10

[0070] Tertiary SSR primers: upstream primer SEQ ID NO.11 and downstream primer SEQ ID NO.12; upstream primer SEQ ID NO.13 and downstream primer SEQ ID NO.14.

[0071] Upstream primer SEQ ID NO.15 and downstream primer SEQ ID NO.16

[0072] Upstream primer SEQ ID NO.17 and downstream primer SEQ ID NO.18

[0073] Upstream primer SEQ ID NO.19 and downstream primer SEQ ID NO.20

[0074] Upstream primer SEQ ID NO.21 and downstream primer SEQ ID NO.22

[0075] Upstream primer SEQ ID NO.23 and downstream primer SEQ ID NO.24

[0076] SSR primary primers are suitable for identifying and distinguishing geographical populations of silkworm germplasm resources (varieties), secondary primers are suitable for identifying and distinguishing different germplasm resource (variety) groups within the same geographical population, and tertiary primers are suitable for identifying and distinguishing all silkworm germplasm resources (varieties) within a germplasm resource (variety) group.

[0077] (3) Electrophoresis to display and differentiate PCR products: The electrophoretic pattern of the PCR product of the genomic DNA of the silkworm germplasm resource (variety) to be identified is compared with the electrophoretic pattern of the amplified product of the corresponding graded identification SSR primers. If the electrophoretic pattern of the PCR product of the silkworm germplasm resource (variety) to be identified matches its unique electrophoretic pattern, then the silkworm germplasm resource (variety) to be identified is the silkworm germplasm resource.

[0078] (variety).

[0079] Furthermore, the electrophoretic patterns specific to the primary SSR primers were selected from one of the four geographical populations: Chinese, Japanese, tropical, and European.

[0080] Furthermore, the electrophoretic pattern specific to the secondary SSR primers is selected from one of the germplasm resources (varieties) groups A, B, or C within the same geographical population.

[0081] Furthermore, the electrophoretic pattern specific to the tertiary SSR primers is selected from one of the smaller number of silkworm germplasm resources (varieties) within the same germplasm resource (variety) group in the same geographical population.

[0082] Preferably, in step (3), the PCR specifically involves: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 40 seconds, annealing at 63°C for 40 seconds, and extension at 72°C for 60 seconds. For the subsequent 15 cycles, the annealing temperature is decreased by 0.5°C per cycle until reaching 56°C. Then, 24 cycles of amplification are performed under the following conditions: denaturation at 94°C for 40 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 60 seconds. After the amplification reaction, the product is extended at 72°C for 5 minutes. After the reaction, the product is stored at 4°C for later use.

[0083] Preferably, in step (3), the PCR reaction system is as follows: 0.5 μl DNA template, 0.5 μl each of forward and reverse primers (10 μmol / L), 2 μl Taq DNA polymerase, 2 μl 2.5 mmol / L dNTPs, and 10× PCR Buffer (Mg2+). 2+ Add 2μl of ultrapure water to bring the total to 20μl.

[0084] More preferably, the total amount of DNA template does not exceed 100 ng.

[0085] Preferably, in step (3), the electrophoresis method is SDS-PAGE gel electrophoresis. After the electrophoretic gel is stained, it is photographed in a chemiluminescence analyzer, and the gel electrophoresis image is saved for analysis.

[0086] More preferably, all the products amplified from the 20 μL PCR reaction system are added to 4 μL of 6× loading buffer, shaken and mixed to serve as the electrophoresis sample, and 5 μL of sample is loaded into each well of the electrophoresis gel.

[0087] More preferably, the separating gel concentration for SDS-PAGE gel electrophoresis is 15%, and the gel is run at 120V for 30 minutes, followed by a run at 150V for 1.5 hours.

[0088] More preferably, the electrophoresis products are displayed using silver staining. The SDS-PAGE gel is placed in a culture dish containing silver staining solution, ensuring the solution covers the gel. The dish is then placed on a shaker at 80-150 rpm for 10-15 minutes. After staining, the gel is rinsed twice with UP water, shaking for 1 minute each time at 150-250 rpm. The rinsed gel is then placed in a culture dish containing developing solution and shaken on a shaker at 80-150 rpm for 10-15 minutes until the bands are clearly visible. The formulation of a 15% separating gel in a 15 mL system is as follows: 7.5 mL of 30% Acr-Bis (29:1), 5.7 mL of 1.5 mol / L Tris-HCl (pH 8.8), 150 μL of 10% SDS, 150 μL of 10% ammonium persulfate, 7 μL of TEMED, and 1.5 mL of ultrapure water.

[0089] Example 1: Screening of candidate SSR primers for identifying silkworm germplasm resources (varieties)

[0090] In order to select candidate SSR primers from the vast silkworm genome database for use in PCR amplification for the identification of silkworm germplasm resources (varieties), the following steps were performed in this embodiment:

[0091] (1) Extracting the whole genome DNA template of silkworms

[0092] (a) Processing of biological samples of silkworm germplasm resources (varieties).

[0093] Fifth-instar larvae of Chinese silkworm variety Su5, Japanese variety Su6, tropical variety Y12-1A, and European variety Fa408 were collected. Two larvae from each of these four geographical populations were randomly selected and combined to form a random sample of eight larvae. Three samples were repeated.

[0094] The steps for extracting larval tissue are as follows:

[0095] First, tie the head and tail of the larvae tightly with a thin thread, immerse them in a 75% ethanol solution for 5 minutes for sterilization, and then wash them twice with physiological saline.

[0096] Next, in a sterile operating table, the larval epidermis is broken open with a scalpel, and the silk gland tissue is extracted with surgical forceps. The extracted silk gland tissue is washed twice with sterilized physiological saline, and the surface moisture of the silk gland is absorbed with sterilized absorbent paper. It is then stored at -80℃ for later use.

[0097] (b) Extract the whole genome DNA of silkworm germplasm resources (varieties) to obtain DNA templates.

[0098] Take an excess of silk gland tissue and place it in a sterilized mortar that has been pre-cooled with liquid nitrogen. Add liquid nitrogen and grind it into powder. Weigh 1000 mg and transfer it into a sterile centrifuge tube.

[0099] Using a commercially available DNA extraction kit, and following the kit's instructions, whole-genome DNA was extracted from silk gland tissue powder in centrifuge tubes, and RNA was further removed. The extracted DNA samples were stored at -20°C.

[0100] (2) PCR reaction to amplify SSR sequence

[0101] SSR primers were designed based on the whole genome sequence of the silkworm from the silkworm genome database. The SSR primers were evenly distributed on 28 linkage groups of the silkworm.

[0102] The SSR sequence of primers for PCR amplification was obtained using the silkworm genomic DNA sample obtained in step (1) as a template.

[0103] The PCR reaction was performed as follows: pre-denaturation at 94℃ for 3 minutes, followed by denaturation at 94℃ for 40 seconds, annealing at 63℃ for 40 seconds, and extension at 72℃ for 60 seconds. For the next 15 cycles, the annealing temperature was decreased by 0.5℃ per cycle until reaching 56℃. Then, 24 cycles of amplification were performed under the following conditions: denaturation at 94℃ for 40 seconds, annealing at 56℃ for 40 seconds, and extension at 72℃ for 60 seconds, followed by a final extension at 72℃ for 5 minutes. After the reaction, the product was stored at 4℃ for later use.

[0104] The PCR reaction system consisted of: 0.5 μl DNA template, 0.5 μl each of forward and reverse primers (10 μmol / L), 2 μl Taq DNA polymerase, 2 μl 2.5 mmol / L dNTPs, 2 μl 10×PCR Buffer (Mg2+ plus), and ultrapure water to a final volume of 20 μl.

[0105] (3) Electrophoresis was used to examine the polymorphic amplification effect of the SSR primers.

[0106] Preferably, PCR products are separated by SDS-PAGE gel electrophoresis, and the polymorphism of the PCR products shown by the electrophoretic bands is observed by photographing the electrophoretic gel after staining.

[0107] The steps for electrophoretic separation of PCR products are as follows:

[0108] First, add all the products amplified from the 20 μL PCR reaction system to 4 μL of 6× loading buffer, vortex to mix, and use as the electrophoresis sample. Add 5 μL to each well of a 21×7 cm 15 mL gel.

[0109] Then, using a 15% separating gel with 1×SDS-tris electrophoresis buffer, the gel was run at 120V for 30 minutes and then at 150V for 1.5 hours.

[0110] After electrophoresis, the SDS-PAGE gel was placed in a culture dish containing silver staining solution, ensuring the solution covered the gel. The gel was then placed on a shaker at 80-150 rpm for 10-15 minutes. After staining, the gel was rinsed twice with UP water, shaking for 1 minute each time at 150-250 rpm. The rinsed gel was then placed in a culture dish containing developing solution and shaken on a shaker at 80-150 rpm for 10-15 minutes, until the bands were clearly visible. The formulation of a 15% separating gel in a 15 mL system is as follows: 7.5 mL of 30% Acr-Bis (29:1), 5.7 mL of 1.5 mol / L Tris-HCl (pH 8.8), 150 μL of 10% SDS, 150 μL of 10% ammonium persulfate, 7 μL of TEMED, and 1.5 mL of ultrapure water.

[0111] Analyze the polymorphism of PCR products and select candidate SSR primers:

[0112] The SDS-PAGE gel was photographed using a chemiluminescence analyzer, and the polymorphism of the PCR products displayed by the electrophoretic bands was observed. If the SSR primers used could amplify polymorphic PCR products from the mixed DNA of the four geographical populations of silkworm, then the SSR primers were selected as candidate SSR primers; if the SSR primers used could not amplify polymorphic PCR products from the mixed DNA of the four geographical populations of silkworm, or if the polymorphic PCR products detected repeatedly were very unstable, then the SSR primers were invalid SSR primers.

[0113] (4) Further, from the above-mentioned candidate SSR primers, SSR primers for graded identification of silkworm germplasm resources (varieties) are selected, with multiple pairs of SSR primers (n<10 pairs) selected from each linkage group. The aforementioned candidate SSR primers for graded identification can generate abundant DNA molecular marker polymorphisms in a large class of silkworm germplasm resources (varieties). The selected SSR primers are evenly distributed across the 28 linkage groups of silkworm, and the selected SSR primers can obtain the genetic differences in molecular evolution among silkworm germplasm resources (varieties) to the greatest extent (see...). Figure 12-14 ).

[0114] Example 2: Screening and Application of SSR Primers for Geographic Population Identification of Silkworm Germplasm Resources (Varieties)

[0115] In order to select the most specific SSR primers for identifying geographical populations of silkworms from the numerous candidate SSR primers for grading and identifying silkworm germplasm resources (varieties) obtained in Example 1, the following steps were performed in this example:

[0116] (1) Following the method in Example 1, the whole genome DNA template of the silkworm was extracted.

[0117] (a) Processing of biological samples of silkworm germplasm resources (varieties).

[0118] One silkworm cocoon (pupa) sample was selected from each of the four geographical populations of silkworm: Chinese, Japanese, tropical, and European.

[0119] Healthy silkworm pupae were extracted from the cocoons. 30 pupae were obtained from each germplasm resource (variety). Every 10 pupae were randomly combined into one random sample, and three biological samples were repeated.

[0120] The steps for extracting pupal tissue are as follows:

[0121] First, tie the head and tail of the pupa tightly with a thin thread, immerse it in a 75% ethanol solution for 5 minutes for sterilization, and then wash it twice with physiological saline.

[0122] Next, in a sterile operating table, the pupal skin is broken open with a scalpel, and the fat body tissue is extracted with surgical forceps. The extracted tissue is washed twice with sterilized physiological saline, and the surface moisture of the fat body is absorbed with sterilized absorbent paper. It is then stored at -80℃ for later use.

[0123] (b) Extract the whole genome DNA of silkworm germplasm resources (varieties) to obtain DNA templates.

[0124] Excess fat body tissue was placed in a sterilized mortar and pre-cooled with liquid nitrogen, and ground into powder with liquid nitrogen. 1000 mg of the powder was then transferred into a sterile centrifuge tube.

[0125] Then, using a commercially available DNA extraction kit, following the kit's instructions, whole-genome DNA was extracted from the fat body sample in the centrifuge tube, and RNA was removed. The extracted DNA sample was stored at -20°C.

[0126] (2) PCR reaction to amplify SSR sequence

[0127] From the candidate SSR primers for the classification and identification of silkworm germplasm resources (variety) obtained in Example 1, one pair of primers was selected for each linkage group, and the SSR sequence was amplified by PCR reaction according to the operation method of step (2) in Example (1).

[0128] (3) Electrophoresis was used to examine the polymorphic amplification effect of the SSR primers.

[0129] Following the procedure in step (3) of Example (1), electrophoresis was performed to examine the polymorphic amplification effect of the SSR primers. If the SSR primers used could amplify a PCR product with abundant polymorphism from the DNA sample of one of the four geographical populations of silkworm, while failing to amplify a PCR product with abundant polymorphism from the DNA samples of the other three geographical populations, then the SSR primers were selected as effective screening SSR primers for geographical populations that could amplify PCR products with abundant polymorphism; if the SSR primers used could amplify PCR products with indistinguishable abundant polymorphism from the DNA samples of more than two of the four geographical populations of silkworm, then the SSR primers were invalid geographical population identification SSR primers.

[0130] (4) Further, a large number of geographical varieties were identified and distinguished from the effective SSR primers selected in step (3) for geographical population identification. The following steps are the operational procedures for identifying and distinguishing 45 silkworm germplasm resources (varieties) of 24 Chinese species and 21 Japanese species:

[0131] First, the 45 silkworm germplasm resources (varieties) to be tested were assigned laboratory numbers, and whole genome DNA templates were extracted from the fat bodies of silkworm pupae according to the method in step (1) of this embodiment.

[0132] Then, referring to the method in step (2) of this embodiment, the SSR sequences of each silkworm germplasm resource (variety) are amplified by PCR reaction;

[0133] Furthermore, referring to the method in step (3) of this embodiment, a single electrophoresis was used to examine the polymorphism of PCR products from all 45 silkworm germplasm resources (varieties);

[0134] Finally, based on the polymorphic consistency of the SSR sequences of all 24 Chinese species or 21 Japanese species amplified by PCR using the various SSR primers used, the preferred primary primers for distinguishing between Chinese and Japanese species were selected (Table 1).

[0135] Figure 1 The results show that all 24 Chinese species detected using the upstream sequence SEQ ID NO.1 and downstream sequence SEQ ID NO.2 (AS0940) of the primary primers in Table 1 exhibit abundant SSR sequence polymorphism. Meanwhile, the SSR sequences of 21 Japanese species did not show any SSR sequence products in the electrophoresis gel regions where the Chinese species exhibited polymorphism. Similarly, Figure 2 The results show that the 21 Japanese species detected using the upstream sequence SEQ ID NO.3 and downstream sequence SEQ ID NO.4 (AS1102) of the primary primers in Table 1 all exhibited abundant SSR sequence polymorphism. Meanwhile, the SSR sequences of the 24 Chinese species showed almost no SSR sequence products in the electrophoresis gel regions where the Japanese species exhibited polymorphism, or the polymorphism of the products was easily distinguishable from that of the Japanese species.

[0136] Therefore, using two sets of paired primers SEQ ID NO.1 / SEQ ID NO.2 (AS0940) and SEQ ID NO.3 / SEQ ID NO.4 (AS1102) for dual identification, it is possible to accurately distinguish 24 Chinese species and 21 Japanese species among 45 silkworm germplasm resources (varieties).

[0137] Example 3: Screening and application of SSR primers for distinguishing germplasm resource (variety) groups of silkworm geographical species in China

[0138] This embodiment uses SSR primers to screen the germplasm resources (varieties) of geographical species to distinguish the specific germplasm resources (varieties) of the 24 Chinese geographical species in Example (2) by using SSR marker technology.

[0139] (1) Extracting the whole genome DNA template of silkworms

[0140] (a) Processing of biological samples of silkworm germplasm resources (varieties).

[0141] Referring to the method in step (4) of Example (2), the 45 silkworm germplasm resources (varieties) to be tested were assigned laboratory numbers.

[0142] Egg samples of the silkworm germplasm resources (varieties) to be identified were incubated at 25℃ (±3℃) and 65-85% relative humidity until the embryos reached the greening stage. Dead and unfertilized eggs were removed, and the greening eggs were collected for surface disinfection. The surface disinfection of the greening eggs involved immersing them in 2-4% (v / v) glutaraldehyde for 15 minutes, followed by washing twice with 100% anhydrous ethanol. After drying the egg surface in a sterile operating table, the eggs were placed at -80℃ for later use.

[0143] (b) Extract the whole genome DNA of silkworm germplasm resources (varieties) to obtain DNA templates.

[0144] First, place the egg sample in a sterilized mortar that has been pre-cooled with liquid nitrogen, add liquid nitrogen and grind it into powder, then take 1000mg and transfer it into a sterile centrifuge tube.

[0145] Furthermore, using a commercially available DNA extraction kit, and following the kit's instructions, whole-genome DNA was extracted from the fat body samples in centrifuge tubes, and RNA was removed. The extracted DNA samples were stored at -20°C.

[0146] (2) PCR reaction to amplify SSR sequence

[0147] From the SSR primers for the graded identification of silkworm germplasm resources (variety) obtained in Example 1, the SSR sequence was amplified by PCR reaction according to the operation method of step (2) in Example (1).

[0148] The candidate SSR primers used for hierarchical identification were selected sequentially from different linkage groups.

[0149] (3) Electrophoresis was used to examine the polymorphic amplification effect of the SSR primers.

[0150] Electrophoresis was performed to examine the polymorphism of the PCR products, following the procedure in step (3) of Example (1).

[0151] SSR primers capable of distinguishing germplasm resource (variety) groups within the same geographical population through PCR products were selected as effective SSR secondary primers (Table 1). In this embodiment, the effective SSR secondary primers were able to accurately distinguish 24 germplasm resources (variety) groups within the Chinese geographical population into different germplasm resource (variety) groups.

[0152] Figure 3 The results show that using the secondary primer pair sequence SEQ ID NO.5 / SEQ ID NO.6 (BS1603) in Table 1, the 24 geographical populations of China can be accurately divided into 3 groups, with the number of germplasm resources (varieties) within each group being 7, 9, and 8, respectively. Using the secondary primer pair sequence SEQ ID NO.7 / SEQ ID NO.8 (BS0905) in Table 1, the 24 geographical populations of China can be accurately divided into 2 groups, with the number of germplasm resources (varieties) within each group being 16 and 8, respectively.

[0153] It is evident that using a single secondary primer or two sets of paired secondary primers in Table 1 for simultaneous dual identification can quickly distinguish the numerous silkworm germplasm resources (varieties) within the geographical populations of China.

[0154] Example 4: Screening and application of SSR primers for differentiating germplasm resource (variety) groups of Japanese geographical silkworm species

[0155] This embodiment uses SSR primers to screen the germplasm resources (varieties) of geographical species to distinguish the specific germplasm resources (varieties) of the 21 Japanese geographical species in Example (2) by using SSR marker technology.

[0156] First, the whole genome DNA template of the silkworm was extracted according to the method of step (1) in Example 3;

[0157] Then, referring to step (2) of Example 3, using the whole genome DNA of the silkworm extracted in the above steps as a template, the SSR sequence was amplified by PCR reaction using the graded identification candidate SSR primers in Table 1.

[0158] Further, referring to step (3) of Example 3, electrophoresis was used to examine the polymorphic amplification effect of the PCR products. SSR primers capable of distinguishing germplasm resource (variety) groups within the same geographical population through PCR products were selected as effective SSR secondary primers (Table 1). In this example, the effective SSR secondary primers were able to accurately distinguish 21 germplasm resources (variety) groups within the Japanese geographical population into different germplasm resource (variety) groups.

[0159] Figure 4The results show that using the secondary primer pair sequence SEQ ID NO.9 / SEQ ID NO.10 (BS1504) in Table 1, the 21 Japanese geographical populations can be accurately divided into 3 groups, with the number of germplasm resources (varieties) in the divided groups being 6, 6, and 9 respectively; using the secondary primer pair sequence SEQ ID NO.11 / SEQ ID NO.12 (BS0305) in Table 1, the 21 Chinese geographical populations can be accurately divided into 2 groups, with the number of germplasm resources (varieties) in the divided groups being 11 and 10 respectively.

[0160] It is evident that using a single secondary primer or two sets of paired secondary primers in Table 1 for simultaneous dual identification can quickly distinguish the numerous silkworm germplasm resources (varieties) within the Japanese geographical population.

[0161] Example 5: Screening and application of SSR primers for identifying germplasm resources (varieties) within the geographical populations of silkworm in China.

[0162] This embodiment further analyzes the polymorphism of multiple germplasm resources (varieties) in the Chinese geographical population of silkworm after grouping the secondary SSR primers in embodiment (3), and screens tertiary SSR primers that can be used to identify silkworm germplasm resources (varieties).

[0163] First, the whole genome DNA template of the silkworm was extracted according to the method of step (1) in Example 3;

[0164] Then, referring to step (2) of Example 3, using the whole genome DNA of the silkworm extracted in the above steps as a template, the SSR sequence was amplified by PCR reaction using the graded identification candidate SSR primers in Table 1.

[0165] Furthermore, referring to step (3) of Example 3, electrophoresis was used to examine the polymorphic amplification effect of the PCR products.

[0166] Select tertiary SSR primers (Table 1) that can identify silkworm germplasm resources (varieties) from a specific germplasm resource (variety) group within the same geographical population using PCR products. In this embodiment, the effective tertiary SSR primers can accurately identify and distinguish multiple germplasm resources (varieties) within the groups after the secondary primer pair sequence SEQ ID NO.5 / SEQ ID NO.6 (BS1603) in Example (3) divides the 24 germplasm resources (varieties) within the Chinese geographical population of silkworm into 3 germplasm resource (variety) groups.

[0167] Figure 5The results show that the tertiary primer pairs in Table 1, SEQ ID NO.13 / SEQ ID NO.14 (CS0203) or SEQ ID NO.15 / SEQ ID NO.16 (CS0601), can identify and distinguish the seven Chinese systematic silkworm germplasm resources (varieties) within the BS1603-A group.

[0168] Figure 6 The results show that using the tertiary primer pairs in Table 1, SEQ ID NO.13 / SEQ ID NO.14 (CS0203) or SEQ ID NO.15 / SEQ ID NO.16 (CS0601), can also identify and distinguish the nine Chinese systematic silkworm germplasm resources (varieties) within the BS1603-B group.

[0169] Figure 7 The results show that using the tertiary primer pairs in Table 1, SEQ ID NO.13 / SEQ ID NO.14 (CS0203) or SEQ ID NO.15 / SEQ ID NO.16 (CS0601), can also identify and distinguish the eight Chinese systematic silkworm germplasm resources (varieties) within the BS1603-C group.

[0170] It is evident that using the tertiary primer pair sequences SEQ ID NO.13 / SEQ ID NO.14 (CS0203) or SEQ ID NO.15 / SEQ ID NO.16 (CS0601) alone or in combination, can quickly and accurately identify multiple germplasm resources (varieties) within the three germplasm resource (varieties) groups after differentiation by the secondary primer pair sequences SEQ ID NO.5 / SEQ ID NO.6 (BS1603) in Example 3, thus achieving the final identification of 24 germplasm resources (varieties) within the Chinese geographical population of silkworm.

[0171] Example 6: Screening and Application of SSR Primers for Identification of Germplasm Resources (Varieties) within Japanese Geographical Populations of Silkworms

[0172] This embodiment further analyzes the polymorphism of multiple germplasm resources (varieties) in the Japanese geographical population of silkworm after grouping the secondary SSR primers in embodiment (3), and screens tertiary SSR primers that can be used to identify silkworm germplasm resources (varieties).

[0173] First, the whole genome DNA template of the silkworm was extracted according to the method of step (1) in Example 3;

[0174] Then, referring to step (2) of Example 3, using the whole genome DNA of the silkworm extracted in the above steps as a template, the SSR sequence was amplified by PCR reaction using the graded identification candidate SSR primers in Table 1.

[0175] Furthermore, referring to step (3) of Example 3, electrophoresis was used to examine the polymorphic amplification effect of the PCR products.

[0176] Select tertiary SSR primers (Table 1) that can identify silkworm germplasm resources (varieties) from a certain germplasm resource (variety) group within the same geographical population using PCR products. In this embodiment, the effective tertiary SSR primers can accurately identify and distinguish multiple germplasm resources (varieties) within the group after the secondary primer pair sequence SEQ ID NO.9 / SEQ ID NO.10 (BS1504) in Example (3) divides 21 germplasm resources (varieties) within the Japanese geographical population of silkworm into 3 germplasm resource (variety) groups.

[0177] Figure 8 The results show that the tertiary primer pairs in Table 1, or SEQ ID NO.15 / SEQ ID NO.16 (CS0601), or SEQ ID NO.17 / SEQ ID NO.18 (CS2708), or SEQ ID NO.19 / SEQ ID NO.20 (CS0901), can all be used to identify and distinguish the six Japanese silkworm germplasm resources (varieties) within the BS1504-A group.

[0178] Figure 9 The results show that using the tertiary primer pairs in Table 1, SEQ ID NO.15 / SEQ ID NO.16 (CS0601), or SEQ ID NO.17 / SEQ ID NO.18 (CS2708), or SEQ ID NO.19 / SEQ ID NO.20 (CS0901), can also identify and distinguish the six Japanese silkworm germplasm resources (varieties) within the BS1504-B group.

[0179] Figure 10 The results show that the tertiary primer pairs in Table 1, namely SEQ ID NO.17 / SEQ ID NO.18 (CS2708), SEQ ID NO.19 / SEQ ID NO.20 (CS0901), or SEQ ID NO.21 / SEQ ID NO.22 (CS1502), can all be used to identify and distinguish the nine Japanese silkworm germplasm resources (varieties) within the BS1504-C group.

[0180] It is evident that using the above-mentioned tertiary primer pairs alone, in combination, or in triplicate, can quickly and accurately identify multiple germplasm resources (varieties) within the three germplasm resource (variety) groups after differentiation by the secondary primer pair sequence SEQ ID NO.9 / SEQ ID NO.10 (BS1504) in Example 3, thus achieving the final identification of 21 germplasm resources (varieties) within the Japanese geographical population of silkworm.

[0181] Example 7: Identification of strains and conservation sites of the same germplasm resource (variety) of silkworm using tertiary SSR primers.

[0182] This embodiment uses the preferred tertiary SSR primers from embodiment (5) for identifying the Su5 variety within the geographical population of silkworms in China, to identify its three strains and preservation locations.

[0183] First, the whole genome DNA template of the silkworm was extracted according to the method of step (1) in Example 3;

[0184] Then, referring to step (2) of Example 3, using the whole genome DNA of the silkworm extracted in the above steps as a template, the SSR sequence was amplified by PCR reaction using the graded identification candidate SSR primers in Table 1.

[0185] Furthermore, referring to step (3) of Example 3, electrophoresis was used to examine the polymorphic amplification effect of the PCR products.

[0186] Figure 11 The results show that using the optimized tertiary SSR primers SEQ ID NO.15 / SEQ ID NO.16 (CS0601) for identifying the Su5 variety within the Chinese geographical population of silkworms in Example (5), or the tertiary primer pair sequences SEQ ID NO.23 / SEQ ID NO.24 (CS0201) in Table 1, the stability and accuracy of DNA molecular markers in three strains of the Chinese Su5 variety—Su5, Su5(Zhen), and Su5(Hu)—and in three preservation units (ZJ, HA, and SH) were tested. This confirms that these two pairs of tertiary SSR primers are effectively applicable to the testing of different samples of the Su5 variety, including samples of different strains of the Su5 variety or samples of the same strain preserved in different preservation units, maintaining a significantly consistent stable molecular marker region (within the dark gray box). Furthermore, Figure 11 The results also revealed significant differences (marked regions within light gray boxes) among the three strains of the Su5 variety or among samples of the same strain from two germplasm resource preservation units, indicating that obvious genetic variation and differentiation have occurred among the strains of the Su5 variety. The same strain of the Su5 variety also showed certain genetic variation or drift after long-term preservation in different units.

[0187] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for graded identification of genetic resources of Chinese and Japanese geographical species of silkworm, characterized in that, Includes the following steps: S1. The DNA of the silkworm to be identified was amplified using primers as shown in SEQ ID NO.1-2. The amplified products were classified once based on their polymorphism to identify the geographical species of China and Japan. S2. Based on the results of the first classification in S1, primers as shown in SEQ ID NO.5-6 were used to perform a second classification of geographical species in China, and primers as shown in SEQ ID NO.9-10 were used to perform a second classification of geographical species in Japan. S3. Based on the results of the secondary classification in S2, the primers shown in SEQ ID NO.13-14 or SEQ ID NO.15-16 are used to classify the geographical species of China in a tertiary manner, and the primers shown in SEQ ID NO.17-18 or SEQ ID NO.19-SEQ ID NO.20 are used to classify the geographical species of Japan in a tertiary manner.

2. The method according to claim 1, characterized in that: The PCR products were displayed by electrophoresis, and the silkworms to be tested were distinguished by comparison with the standard electrophoresis pattern.

3. The method according to claim 1, characterized in that: The electrophoretic products were visualized using silver staining.

4. The method according to claim 1, characterized in that: The samples from which the DNA of the silkworm to be tested was extracted were selected from eggs, larvae, pupae, or adults.

5. The method according to claim 1, characterized in that: The amplification reaction system includes a DNA template, amplification primers, Taq DNA polymerase, and dNTPs.

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