SNP Molecular Markers Related to Bombyx mori Nucleopolyhedrovirus Resistance and Their Applications

Abstract

The present invention belongs to the technical fields of molecular biology and molecular breeding, and particularly relates to SNP molecular markers related to Bombyx mori nucleopolyhedrovirus (BmNPV) resistance and their applications. The SNP molecular markers include the combination of the Chr3-746 marker and the Chr27-5071 marker; the Chr3-746 marker is located at the 14951064th position of the 3rd chromosome sequence of the Bombyx mori p50T genome, and the base polymorphism is G / A; the Chr27-5071 marker is located at the 9462596th position of the 27th chromosome sequence of the Bombyx mori p50T genome, and the base polymorphism is C / G. The SNP molecular markers of the present invention can effectively distinguish BmNPV-resistant and -susceptible Bombyx mori, reduce the false positive rate of detection, improve the accuracy rate, and can be screened quickly and in a high-throughput manner, solve the technical problems of traditional Bombyx mori breeding, and accelerate the breeding of BmNPV-resistant Bombyx mori varieties.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and molecular breeding, and particularly relates to SNP molecular markers related to resistance to Bombyx mori nucleopolyhedrovirus and their applications. Background Art

[0002] The silkworm is an important economic insect for producing silk cocoons. Blood septicemia caused by Bombyx mori nucleopolyhedrovirus (BmNPV) is a major infectious disease in the world's sericulture industry. The loss of silk cocoons caused by this disease accounts for more than 60% of the total loss caused by silkworm diseases. Existing research has proved that there are major genes controlling the resistance of silkworms to BmNPV disease; experimental studies on the resistance of silkworms to BmNPV and its inheritance pattern have shown that there are differences in resistance among silkworm strains, the resistance is incompletely dominant over susceptibility, and the resistance is controlled by more than two pairs of genes. For the prevention and control of BmNPV disease in silkworms, breeding new BmNPV-resistant varieties is the most direct and effective way. Many studies at home and abroad have been committed to the research of new resistant silkworm varieties, and the identification of their resistance genes plays an important role in the breeding and popularization of disease-resistant varieties.

[0003] Marker assisted selection (MAS) is a technique that uses markers related to traits (such as DNA variations) to indirectly select target genes or target traits, including RAPD, RFLP, SSR, and SNP, etc.

[0004] Single-nucleotide polymorphism (SNP) refers to a DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level, including base transitions and transversions. With the development of high-throughput sequencing technology, SNP sites have been widely discovered in the genome. As the third-generation molecular marker technology, SNP has the characteristics of a large number, uniform distribution in the genome; high genetic stability and low mutation frequency; easy to detect, suitable for rapid and large-scale screening, etc., and is widely used in genetic map construction, gene mapping, assisted breeding, etc. Kompetitive Allele Specific PCR (KASP) is a commonly used high-throughput genotyping technology for SNP marker detection. It genotypes SNPs based on the specific matching of the base at the primer end, that is, detects the target SNP marker through two site-specific upstream primers, one common downstream primer, and two common fluorescent probes. The KASP technology has high stability and accuracy for SNP marker detection, and can achieve high-throughput screening, which is the main technical means for molecular assisted breeding and identification of target traits.

[0005] Most of the molecular markers linked to the genes related to Bombyx mori nucleopolyhedrovirus (BmNPV) disease resistance discovered in current research are RAPD markers or SSR markers. However, the identification operations of these two types of molecular markers for resistance genes are complex, the detection efficiency is low, and they show incomplete linkage with resistance traits, making them unsuitable for molecular marker-assisted breeding of B. mori.

[0006] Research based on genetic laws has shown that the BmNPV disease resistance trait in B. mori is controlled by two or more pairs of genes and there is an additive effect; existing research results have also shown that molecular markers linked to a single resistance gene cannot effectively identify the BmNPV disease resistance trait in B. mori. At present, the breeding of BmNPV disease-resistant varieties in B. mori mainly relies on traditional phenotypic identification methods, which are labor-intensive, have a long cycle, and the accuracy is easily affected by the environment. Therefore, developing SNP molecular markers applicable to high-throughput detection platforms and capable of quickly and accurately identifying the BmNPV disease resistance trait in B. mori is of great significance for popularizing the application of molecular marker technology and improving the efficiency and technical level of B. mori breeding. Summary of the Invention

[0007] To solve the technical problems in the above background, the present invention provides a set of SNP molecular markers for BmNPV resistance in B. mori. Based on the SNP molecular marker set and combined with the KASP detection technology, effective, rapid, and accurate identification of BmNPV-resistant varieties in B. mori can be achieved.

[0008] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0009] The present invention first provides a set of SNP molecular markers for BmNPV resistance in B. mori, including the Chr3-746 marker located on chromosome 3 of B. mori and the Chr27-5071 marker located on chromosome 27 of B. mori.

[0010] Furthermore, the genetic distance of the Chr3-746 marker on chromosome 3 is 12.5 cM, and the interval for mapping the resistance gene on chromosome 3 is 10.6 cM to 14.7 cM; the genetic distance of the Chr27-5071 marker on chromosome 27 is 13.8 cM, and the interval for mapping the resistance gene on chromosome 27 is 12.1 cM to 15.8 cM.

[0011] The Chr3-746 marker is located at the 14951064th position of the chromosome 3 sequence of the B. mori p50T genome, with a base polymorphism of G / A, and the A allele is closely linked to the BmNPV disease resistance gene in B. mori; the Chr27-5071 marker is located at the 9462596th position of the chromosome 27 sequence of the B. mori p50T genome, with a base polymorphism of C / G, and the G allele is closely linked to the BmNPV disease resistance gene in B. mori.

[0012] The present invention also provides primers for detecting the SNP molecular marker. The primer combination for detecting the above SNP molecular marker is a KASP primer combination. Each KASP primer combination consists of two allele-specific forward primers F1 and F2 with different terminal bases and one reverse universal primer R.

[0013] Furthermore, the primers include primer sets for amplifying the Chr3-746 marker and for amplifying the Chr27-5071 marker. Among them, the primer set for amplifying the Chr3-746 marker includes the primer sequences shown in SEQ.ID.NO.1-3, and the primer set for amplifying the Chr27-5071 marker includes the primer sequences shown in SEQ.ID.NO.4-6.

[0014] Furthermore, in the primer set for amplifying the Chr3-746 marker, the primer shown in SEQ.ID.NO.1 is used to match the A allele, and the primer shown in SEQ.ID.NO.2 is used to match the G allele; in the primer set for amplifying the Chr27-5071 marker, the primer shown in SEQ.ID.NO.4 is used to match the G allele, and the primer shown in SEQ.ID.NO.5 is used to match the C allele.

[0015] Furthermore, the primer shown in SEQ.ID.NO.1 in the primer set for amplifying the Chr3-746 marker and the primer shown in SEQ.ID.NO.4 in the primer set for amplifying the Chr27-5071 marker are respectively labeled with HEX or VIC fluorescent probes; the primer shown in SEQ.ID.NO.2 in the primer set for amplifying the Chr3-746 marker and the primer shown in SEQ.ID.NO.5 in the primer set for amplifying the Chr27-5071 marker are respectively labeled with FAM fluorescent probes.

[0016] Specifically, the primer information is as follows:

[0017] Primer set for amplifying the Chr3-746 marker:

[0018] F1(SEQ.ID.NO.1): 5’-gaaggtcggagtcaacggattGCATGCTTTAAGTCAAAACGCATTAA-3’

[0019] (For matching the A allele, the lowercase letter part is the HEX fluorescent tag sequence);

[0020] F2 (SEQ.ID.NO.2): 5’-gaaggtgaccaagttcatgctGCATGCTTTAAGTCAAAACGCATTAG-3’ (for matching the G allele, the lowercase part is the FAM fluorescent tag sequence);

[0021] R (SEQ.ID.NO.3): 5’-GAATCCTATTCACAATGCAGGGTG-3’.

[0022] Primer set for amplifying the Chr27 - 5071 marker:

[0023] F1 (SEQ.ID.NO.4): 5’-gaaggtcggagtcaacggattGAATCGAGCCCACGACCTTG-3’ (for matching the G allele, the lowercase part is the HEX fluorescent tag sequence);

[0024] F2 (SEQ.ID.NO.5): 5’-gaaggtgaccaagttcatgctGAATCGAGCCCACGACCTTC-3’ (for matching the C allele, the lowercase part is the FAM fluorescent tag sequence);

[0025] R (SEQ.ID.NO.6): 5’-GTAATAGTGCACGGTAGAATGACG-3’.

[0026] The present invention also provides products for detecting or assisting in detecting the resistance of silkworm to BmNPV, including detection reagents, reagent kits, chips, etc.; the products include SNP molecular markers for silkworm BmNPV resistance or primers for detecting SNP molecular markers for silkworm BmNPV resistance.

[0027] The present invention also provides a method for developing SNP molecular markers related to the resistance of silkworm to BmNPV disease, comprising the following steps:

[0028] S1. Construct a genetic mapping population using the parental materials of resistant and susceptible silkworm to BmNPV disease;

[0029] S2. Extract the genomic DNA of the parental materials of resistant and susceptible materials and the mapping population in step S1, and develop SNP markers between the parents through reduced-representation genome sequencing (RAD-Seq);

[0030] S3. Screen the SNP markers obtained in step S2 to construct a genetic map;

[0031] S4. Use the phenotypic data of the mapping population in step S1 combined with the genetic map constructed in step S3 for QTL interval mapping;

[0032] S5. Screen SNP molecular markers linked to resistance genes through the LOD peak in step S4;

[0033] S6. Design KASP primers for the SNP molecular markers screened in step S5;

[0034] S7. Use the kit containing the KASP primers in step S6 and adopt the KASP method to verify the SNP molecular markers.

[0035] The present invention also provides the application of the SNP molecular markers, or the primers, or the products, or the detection methods in any of the following aspects:

[0036] (1) Identification of silkworm germplasm resources or varieties;

[0037] (2) Identification of the purity of silkworm germplasm resources or varieties;

[0038] (3) Identification of the BmNPV disease resistance traits of silkworms;

[0039] (4) Molecular marker-assisted breeding of silkworms;

[0040] (5) Mapping and identification of BmNPV disease resistance genes in silkworms;

[0041] (6) Development of products for identifying silkworm germplasm resources or traits.

[0042] According to the above applications, preferably, the above applications include the following steps:

[0043] Extract the genomic DNA of the silkworm sample to be tested; detect the gene polymorphisms or genotypes of the Chr3-746 marker and the Chr27-5071 marker for the extracted genomic DNA; make judgments, identifications, or assist breeding according to the detection results.

[0044] Among them, the detection is carried out using the KASP technology, and the A allele of the Chr3-746 marker and the G allele of the Chr27-5071 marker are respectively labeled with HEX or VIC fluorescent probes; the G allele of the Chr3-746 marker and the C allele of the Chr27-5071 marker are respectively labeled with FAM fluorescent probes.

[0045] Only when HEX or VIC fluorescence signals are simultaneously detected for both the Chr3-746 marker and the Chr27-5071 marker, that is, the genotype of the Chr3-746 marker is AA or AG genotype and the genotype of the Chr27-5071 marker is GG or GC genotype, can it be determined that the tested silkworm is a BmNPV disease-resistant silkworm.

[0046] The method for judging, identifying or assisting in breeding based on the detection results is as follows: If the genotype of the Chr3-746 marker is GG or the genotype of the Chr27-5071 marker is CC, the silkworm to be detected is a silkworm susceptible to BmNPV disease; if the genotype of the Chr3-746 marker is AA or AG and the genotype of the Chr27-5071 marker is GG or GC, the silkworm to be detected is a silkworm resistant to BmNPV disease.

[0047] The PCR amplification system of the KASP technology is as follows for a 5 μL reaction: 2.5 μL KASP Master Mix (2x), 0.7 μL KASP primer set (0.15 μL each of F1 and F2 primers at a concentration of 10 μM, 0.4 μL of R primer at a concentration of 10 μM), 1 μL genomic DNA (20 - 50 ng / μL), and ddH2O is added to make up the system to 5 μL.

[0048] The PCR amplification program of the KASP technology is: pre-denaturation at 94 °C for 10 min; denaturation at 94 °C for 20 s, annealing at 65 °C for 1 min, with the annealing temperature decreasing by 0.8 °C for each cycle, for a total of 10 cycles; denaturation at 94 °C for 20 s, annealing at 57 °C for 1 min, for a total of 27 - 30 cycles; extension at 25 °C for 30 s, and fluorescence signals are collected.

[0049] Preferably, the detected silkworms with the genotype of AA or AG for the above-mentioned Chr3-746 marker and the genotype of GG or GC for the Chr27-5071 marker are selected as parents for breeding.

[0050] The beneficial effects of the present invention:

[0051] The present invention hybridizes a BmNPV-resistant silkworm strain and a BmNPV-susceptible silkworm strain to obtain the F1 generation, and the F1 generation is self-crossed to obtain the F2 generation recombinant inbred lines. A genetic mapping population consisting of 152 individuals is formed. The BmNPV disease phenotypes of the F2 generation genetic population are identified as resistant and susceptible populations. Genomic DNA of the parents and individuals of the genetic population is extracted for reduced-representation genomic sequencing (RAD-Seq) to develop polymorphic markers, and then a genetic linkage map is constructed using 3060 SNP markers developed. Through the resistant and susceptible populations, combined with the high-density linkage map, QTL analysis is performed on the traits, and the Chr3-746 marker closely linked to the QTL gene on chromosome 3 and the Chr27-5071 marker closely linked to the QTL gene on chromosome 27 are obtained. The detection using the double markers of Chr3-746 and Chr27-5071 can effectively identify individuals with disease-resistant traits and reduce the false positive rate of single marker detection.

[0052] The SNP molecular markers for Bombyx mori BmNPV disease resistance developed in the present invention can effectively identify the resistance traits of Bombyx mori to BmNPV disease, and are not affected by factors such as time, sampling location, and environment. They can be applied to assist in the breeding of Bombyx mori and accelerate the breeding of Bombyx mori varieties resistant to BmNPV disease.

[0053] The present invention detects the above SNP molecular markers by KASP genotyping detection technology. By using the characteristics of high sensitivity, high throughput, and easy operation of KASP technology, it can quickly, accurately, and with high throughput detect and screen Bombyx mori varieties resistant to BmNPV disease, improving the breeding efficiency.

[0054] By developing the above SNP molecular marker set, the present invention discovers that there are major resistance genes on chromosome 3 and chromosome 27 of Bombyx mori, which has great reference value for the mapping and identification of Bombyx mori BmNPV disease resistance genes and the development of other Bombyx mori BmNPV disease resistance molecular markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a schematic diagram of the development process of the SNP molecular marker set for Bombyx mori BmNPV disease resistance.

[0056] Figure 2 It is a schematic diagram of the construction of a Bombyx mori genetic mapping population.

[0057] Figure 3 It is a Bombyx mori genetic linkage map.

[0058] Figure 4 It is the result of the association analysis of Bombyx mori BmNPV disease resistance traits.

[0059] Figure 5 It is a schematic diagram of the localization of the SNP molecular marker set on the Bombyx mori genome chromosome, showing the genetic distance of the Chr3-746 marker on chromosome 3 of Bombyx mori and the disease-resistant QTL interval on chromosome 3, as well as the genetic distance of the Chr27-5071 marker on chromosome 27 of Bombyx mori and the disease-resistant QTL interval on chromosome 27.

[0060] Figure 6 It is a genotype map of the Chr3-746 marker in the Bombyx mori F2 generation genetic population. The AA and AG genotypes are the homozygous and heterozygous types containing the resistance gene on chromosome 3, and GG is the reference genotype (susceptible homozygous type).

[0061] Figure 7 It is a genotype map of the Chr27-5071 marker in the Bombyx mori F2 generation genetic population. The GG and GC genotypes are the homozygous and heterozygous types containing the resistance gene on chromosome 27, and CC is the reference genotype (susceptible homozygous type).

[0062] Figure 8Schematic diagram of the molecular marker-assisted selection breeding process Detailed implementation mode

[0063] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the protection scope of the present invention

[0064] Unless otherwise specified, the following experimental methods are all conventional methods in the technical field; unless otherwise specified, the materials and reagents used in the following experiments are all commercially available products

[0065] The silkworm varieties used are the resistant strain 'NB' and the susceptible strain '306', both of which are publicly known varieties and are preserved in the laboratory of the School of Life Sciences, Jiangsu University

[0066] Example 1. Development of a SNP molecular marker group resistant to BmNPV disease in silkworms

[0067] The development process of the SNP molecular marker group resistant to BmNPV disease in silkworms is as Figure 1 , and a SNP molecular marker group including the Chr3-746 marker and the Chr27-5071 marker is obtained through screening and verification. The specific steps are as follows

[0068] (1) Construction of a genetic mapping population

[0069] Using the BmNPV-resistant silkworm strain 'NB' as the male parent and the BmNPV-susceptible silkworm strain '306' as the female parent to hybridize to obtain the F1 generation, and backcrossing the F1 generation with the silkworm strain '306' to obtain the BC1 generation. There are a total of 152 progeny individuals in the backcross line. At the 3rd instar stage, the BC1 generation population is divided into a disease-resistant group (surviving after virus feeding) and a disease-susceptible group (dying after virus feeding) by feeding NPV virus at a concentration of 10 8 BmNPV / mL( Figure 2 );

[0070] (2) Development of SNP markers by reduced-representation genome sequencing

[0071] Using the phenol-chloroform extraction method to extract the genomic DNA of a total of 154 individuals, including the parents 'NB' and '306' and the recombinant inbred lines

[0072] The 154 individuals mentioned above were subjected to reduced-representation genome sequencing (RAD-Seq). By aligning with the silkworm reference genome Bombyx mori (http: / / silkbase.ab.a.u-tokyo.ac.jp / cgi-bin / index.cgi), based on the parental genotyping results, polymorphic markers between parents were developed; sites with missing parental information were filtered, and parental polymorphic sites conforming to the mapping marker types of this population were screened. For the BC1 population, polymorphic sites with homozygous differences between the parents were screened (for example: for a certain SNP site, the genotype of parent 1 is CC, the genotype of parent 2 is AA, and the parental genotypes are both homozygous and different between the parents). A total of 3,060 SNP markers were obtained.

[0073] (3) Construction of genetic linkage map

[0074] The offspring population was genotyped using the SNP molecular markers screened above and genotype coding was performed, and information such as the number of individuals with abnormal bases at each locus, the number of individuals with missing offspring genotyping, and the segregation distortion chi-square value were calculated. The coded offspring genotyping results were imported into the joinmap5 software for map construction. To ensure the map quality, the following conditions were filtered using joinmap5:

[0075] a. The number of individuals with abnormal bases at the locus is 0;

[0076] b. The number of individuals with missing offspring genotyping < 20%;

[0077] After the filtration was completed, linkage group division was performed, and the LOD value was set to 2 - 10; each linkage group was sorted using the regression algorithm; the Kosambi function was used to convert the recombination rate into genetic distance.

[0078] (4) QTL mapping analysis and screening of resistance SNP molecular markers

[0079] Using the disease-resistant and disease-susceptible phenotype data of the mapping population combined with the linkage map obtained in step (3) ( Figure 3 ), QTL analysis was performed on the traits using the MapQTL6 software. The specific analysis steps are as follows:

[0080] a. Using permutation tests with 1,000 repetitions, estimate the LOD thresholds at the α = 0.05 level for individual linkage groups and the genome-wide range;

[0081] b. Perform QTL analysis using the interval mapping method (Interval mapping, IM), scanning the possibility of QTL existence every 1 cM on each linkage group;

[0082] c. Using the LOD value at the α = 0.05 level within a single linkage group as the threshold, that is, when a LOD peak value is greater than or equal to the threshold, it is considered that there is a QTL at this locus, and the position of the LOD peak is the most likely position of the corresponding QTL gene.

[0083] Using plink (version: 1.07) software, an association analysis of 3060 SNP markers on the linkage map with the resistance trait of Bombyx mori to BmNPV was carried out for each SNP marker, and the results are shown in Figure 4 .

[0084] The QTL analysis results of the traits showed that under the condition of setting the threshold LOD ≥ 4, only linkage group 3 (chromosome 3) and linkage group 27 (chromosome 27) showed LOD peak values significantly greater than the threshold, that is, there were obvious QTL (BmNPV disease resistance) genes on chromosome 3 and chromosome 27 respectively. Among them, the higher the LOD peak value, the closer the linkage between the corresponding SNP molecular marker and the QTL gene.

[0085] The SNP molecular markers corresponding to the highest LOD peak positions on chromosome 3 and chromosome 27 were selected respectively. Finally, 2 SNP markers linked to the resistance gene were screened and named Chr3-746 and Chr27-5071. The genetic distance of the Chr3-746 marker on chromosome 3 was 12.5 cM, and the genetic distance interval of the resistance gene on chromosome 3 was obtained through the 2-LOD confidence interval as 10.6 cM - 14.7 cM ( Figure 5 ); the genetic distance of the Chr27-5071 marker on chromosome 27 was 13.8 cM, and the genetic distance interval of the resistance gene on chromosome 27 was obtained through the 2-LOD confidence interval as 12.1 cM - 15.8 cM ( Figure 5 ); corresponding to the Bombyx mori p50T genome, the Chr3-746 marker was located at the 14951064th position of the chromosome 3 sequence, and the base polymorphism was G / A, among which the A allele was closely linked to the Bombyx mori BmNPV disease resistance gene; the Chr27-5071 marker was located at the 9462596th position of the chromosome 27 sequence, and the base polymorphism was C / G, among which the G allele was closely linked to the Bombyx mori BmNPV disease resistance gene.

[0086] The QTL analysis results showed that the LOD value of the Chr3-746 marker was 22.42, and the contribution rate to the phenotype was 49.8%; the LOD value of the Chr27-5071 marker was 17.38, and the contribution rate to the phenotype was 41.4%; so the Chr3-746 marker and the Chr27-5071 marker were highly associated with the resistance trait of Bombyx mori to BmNPV and could be used as SNP molecular markers for the resistance of Bombyx mori to BmNPV.

[0087] (5) KASP Primer Design

[0088] According to the Bombyx mori reference genome

[0089] sequences of Bombyx mori (http: / / silkbase.ab.a.u-tokyo.ac.jp / cgi-bin / index.cgi), the flanking sequences of 150 bp on each side of the Chr3-746 marker and the Chr27-5071 marker were extracted respectively. Preferably, the KASP primer combination corresponding to the SNP marker was designed using Primer5.0 software.

[0090] The KASP primer combinations for detecting the genotypes of the Chr3-746 marker and the Chr27-5071 marker are shown in Table 1 specifically.

[0091] Table 1. KASP Primer Combinations

[0092]

[0093] (6) KASP Verification of SNP Molecular Markers

[0094] Using the BC1 generation trait-segregating population constructed with the 'NB' resistant parent and the '306' susceptible parent (after feeding BmNPV, the surviving individuals were divided into the resistant group and the dead individuals were divided into the susceptible group) as the experimental material, genomic DNA was extracted as the template, and 1 blank control (adding water as NTC) was set, with a total of 156 samples.

[0095] Fluorescent labels were added to the primers for amplifying the two markers:

[0096] Primer set for amplifying the Chr3-746 marker:

[0097] F1 (SEQ.ID.NO.1): 5’-gaaggtcggagtcaacggattGCATGCTTTAAGTCAAAACGCATTAA-3’ (for matching the A allele, the lowercase part is the HEX fluorescent label sequence);

[0098] F2 (SEQ.ID.NO.2): 5’-gaaggtgaccaagttcatgctGCATGCTTTAAGTCAAAACGCATTAG-3’ (for matching the G allele, the lowercase part is the FAM fluorescent label sequence);

[0099] R (SEQ.ID.NO.3): 5’-GAATCCTATTCACAATGCAGGGTG-3’.

[0100] Primer set for amplifying Chr27-5071 marker:

[0101] F1 (SEQ.ID.NO.4): 5’-gaaggtcggagtcaacggattGAATCGAGCCCACGACCTTG-3’ (for matching G allele, the lowercase part is the HEX fluorescent tag sequence);

[0102] F2 (SEQ.ID.NO.5): 5’-gaaggtgaccaagttcatgctGAATCGAGCCCACGACCTTC-3’ (for matching C allele, the lowercase part is the FAM fluorescent tag sequence);

[0103] R (SEQ.ID.NO.6): 5’-GTAATAGTGCACGGTAGAATGACG-3’.

[0104] Add KASP primers and KASP Master Mix (Low Rox) from LGC company, and perform PCR amplification, fluorescence scanning and data analysis on the ABI QuantStudio6 (Q6) fluorescence quantitative platform. Preferably, the PCR amplification system is shown in Table 2.

[0105] Table 2. PCR amplification system

[0106] Component Volume (μL) 2×KASP Master Mix 2.5 F1 primer (10μM) 0.15 F2 primer (10μM) 0.15 R primer (10μM) 0.4 DNA template (20~50ng / μL) 1 <![CDATA[H2O]]> up to 5

[0107] Preferably, the fluorescence quantitative PCR reaction conditions are: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 20 s, annealing at 65°C for 1 min, with the annealing temperature decreasing by 0.8°C for each cycle, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing at 57°C for 1 min, for a total of 27 - 30 cycles; extension at 25°C for 30 s, and collect fluorescence signals.

[0108] (7) Result analysis

[0109] After fluorescence collection, data analysis is performed by the Q6 platform software to obtain the genotyping results of Chr3-746 marker and Chr27-5071 marker (the genotyping maps are shown in Figure 6 、 Figure 7), where the allelic genotypes closely linked to the HEX fluorescently labeled resistance gene. The detection of the HEX fluorescent signal indicates the presence of the resistance gene in the tested individual. HEX / HEX represents the resistant homozygous type, HEX / FAM represents the resistant heterozygous type, and FAM / FAM represents the susceptible homozygous type; for the Chr3-746 marker, HEX / HEX is the AA genotype, HEX / FAM is the AG genotype, and FAM / FAM is the GG genotype; for the Chr27-5071 marker, HEX / HEX is the GG genotype, HEX / FAM is the GC genotype, and FAM / FAM is the CC genotype.

[0110] The genotypes of the Chr3-746 marker and the Chr27-5071 marker in the BmNPV disease-resistant population and the BmNPV disease-susceptible population were respectively counted, and the chi-square test was used to verify the significance of the association between the SNP molecular marker and the trait. The results are shown in Table 3. According to Table 3, the p-values of the chi-square tests for the Chr3-746 marker and the Chr27-5071 marker are both less than 0.001, showing extremely significant association with the BmNPV disease resistance trait of silkworms.

[0111] Table 3. Chi-square tests of the Chr3-746 marker and the Chr27-5071 marker

[0112]

[0113] Combined with the phenotypic data of the silkworm genetic population, the detection of the A allele closely linked to the resistance gene of the Chr3-746 marker and the G allele closely linked to the resistance gene of the Chr27-5071 marker in the BmNPV disease-resistant population and the susceptible population of silkworms was counted. At the same time, the known resistance RAPD marker AY380833.1 (GeneBank) of silkworms was used to detect the resistant and susceptible populations and count the results.

[0114] From the statistical results of the disease-resistant population, it can be seen that more than 92% of the disease-resistant silkworms simultaneously detected the A allele of the Chr3-746 marker and the G allele of the Chr27-5071 marker (Tables 4 and 6); from the statistical results of the disease-susceptible population, it can be seen that more than 30% of the disease-susceptible silkworms detected the resistance allele of the Chr3-746 marker or the Chr27-5071 marker or the AY380833.1 marker, but the probability of simultaneously detecting the A allele of the Chr3-746 marker and the G allele of the Chr27-5071 marker was less than 4% (Tables 5 and 6), indicating that the SNP molecular marker group composed of the Chr3-746 marker and the Chr27-5071 marker can effectively distinguish between BmNPV-resistant silkworms and BmNPV-susceptible silkworms in the identification of resistance traits. Its effective detection rate for disease-resistant silkworms is more than 92%, and the effective detection rate for disease-susceptible silkworms is more than 96%. Therefore, the SNP molecular marker group composed of the Chr3-746 marker and the Chr27-5071 marker can effectively identify BmNPV-resistant individuals of silkworms and can be applied to the identification of silkworm germplasm resources and the assistance of silkworm breeding.

[0115] Table 4. Detection of molecular markers in the BmNPV-resistant population of silkworms

[0116]

[0117] Note: The amplified fragment size of the RAPD marker AY380833.1 in disease-resistant individuals is 736 bp, and the amplified fragment size in disease-susceptible individuals is 1628 bp. Amplifying the 736 bp fragment represents the presence of the resistance gene; the genotypes AA or AG of the Chr3-746 marker are the homozygous or heterozygous types of the A allele closely linked to the resistance gene, representing the presence of the resistance gene on chromosome 3; the genotypes GG or GC of the Chr27-5071 marker are the homozygous or heterozygous types of the G allele closely linked to the resistance gene, representing the presence of the resistance gene on chromosome 27; the genotype combination (AA or AG)+(GG or GC) of the Chr3-746+Chr27-5071 marker represents the presence of both the resistance gene on chromosome 3 and the resistance gene on chromosome 27.

[0118] Table 5. Detection of molecular markers in the BmNPV-susceptible population of silkworms

[0119]

[0120] Note: For the RAPD marker AY380833.1, the amplified fragment size is 736 bp in disease-resistant individuals and 1628 bp in disease-susceptible individuals. The amplification of the 736-bp fragment indicates the presence of the resistance gene; for the Chr3-746 marker, the genotypes AA or AG represent the homozygous or heterozygous form of the A allele tightly linked to the resistance gene, indicating the presence of the resistance gene on chromosome 3; for the Chr27-5071 marker, the genotypes GG or GC represent the homozygous or heterozygous form of the G allele tightly linked to the resistance gene, indicating the presence of the resistance gene on chromosome 27; the combined genotype (AA or AG)+(GG or GC) of the Chr3-746+Chr27-5071 marker combination indicates the presence of the resistance gene on both chromosome 3 and chromosome 27.

[0121] Table 6. Corresponding results of molecular marker genotypes and known phenotypes of samples in the BC1 generation trait segregation population

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Note: The underlines indicate the cases where the combined genotypes of the Chr3-746 marker and the Chr27-5071 marker do not match the individual phenotypes.

[0129] In this example, by detecting the known BmNPV resistance RAPD marker AY380833.1 (GeneBank) in silkworms as a comparison, the accuracy rate of detecting resistant silkworms using the SNP molecular marker group composed of the Chr3-746 marker and the Chr27-5071 marker is over 92% (Table 4), the false positive rate for detecting susceptible silkworms is below 4% (Table 5), and the accuracy rate for identifying the resistant and susceptible phenotypes of silkworms is 94.86%. Compared with the 75.43% identification accuracy rate of the AY380833.1 marker, it is increased by 19.43% (Table 6); at the same time, by comparing the detection results of the single markers Chr3-746, Chr27-5071, or AY380833.1 in the susceptible population, it is found that the single marker detection has a false positive rate of over 30%; it can be seen that the detection of the SNP molecular marker group described in the present invention can greatly reduce the false positive rate of detection, and the effective identification of susceptible individuals reaches over 96% (Table 5).

[0130] Example 2. Application of the SNP molecular marker group resistant to Bombyx mori nucleopolyhedrovirus (BmNPV) in the breeding of BmNPV-resistant strains of Bombyx mori

[0131] (1) The process of marker-assisted selection is shown in Figure 8 as follows:

[0132] Using the wings of the moths of the 'NB' strain as materials, genomic DNA was extracted, and the genotypes of the Chr3-746 marker and the Chr27-5071 marker were detected by the KASP method. The moths with the AA genotype of the Chr3-746 marker and the GG genotype of the Chr27-5071 marker were selected as male parents, and the productive varieties with excellent economic traits were selected as female parents for hybridization. Then, they were continuously backcrossed with the recurrent parent with excellent economic traits to the 8th generation, and then self-crossed for more than 2 generations to select the moth areas with non-segregating resistant traits for subculture. To ensure the accurate transmission of the resistance gene without loss and also consider the simplicity of detection, the SNP molecular marker group was used for selection every 2 generations of backcrossing. The moth area rearing - sampling detection method was adopted. For the individuals in the moth area that could simultaneously detect the A allele of the Chr3-746 marker and the G allele of the Chr27-5071 marker, this moth area was subcultured. The subcultured moth areas of BC8 were self-crossed and intensively selected 2 times, and the offspring with homozygous BmNPV-resistant genotypes were obtained through KASP and virus challenge screening.

[0133] (2) Comparison of marker-assisted selection technology with traditional breeding of disease-resistant Bombyx mori

[0134] Breeding experiments were carried out under the same time and the same environment. Through traditional virus addition selection breeding as a comparison, after 11 generations of selection, 2 BmNPV-resistant strains of Bombyx mori were obtained, namely the resistant strain 'NB-R1' obtained by traditional virus addition selection breeding and the resistant strain 'NB-R2' obtained by marker-assisted selection technology breeding.

[0135] Compare the disease resistance effects of traditional virus addition selection and marker-assisted selection:

[0136] For the 'NB-R1' strain and the 'NB-R2' strain, oral virus addition identification was carried out with 3.2×10 8 BmNPV / mL, and the average mortality was used as the criterion for judging resistance. The results are shown in Table 7. In Table 7, the average mortality of the 'NB-R1' strain obtained by traditional breeding was 13.3%, while the average mortality of the 'NB-R2' strain selected by molecular markers was only 2.8%, and the selection effect was very ideal.

[0137] In this example, compared with the traditional breeding method, the survival rate of the resistant silkworms obtained by the combined application of the SNP molecular markers developed in the present invention in the breeding of disease-resistant silkworms was increased by more than 20% (Table 7).

[0138] Table 7. Comparison of breeding effects between traditional silkworm disease-resistant breeding and molecular marker-assisted selection technology breeding

[0139]

[0140] The present invention can be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the above-disclosed embodiments are only illustrative and not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention. Sequence Listing <110> Jiangsu University <120> SNP molecular markers related to Bombyx mori nucleopolyhedrovirus resistance and their applications <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 47 <212> DNA <213> Artificial Sequence <400> 1 gaaggtcgga gtcaacggat tgcatgcttt aagtcaaaac gcattaa 47 <210> 2 <211> 47 <212> DNA <213> Artificial Sequence <400> 2 gaaggtgacc aagttcatgc tgcatgcttt aagtcaaaac gcattag 47 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 gaatcctatt cacaatgcag ggtg 24 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <400> 4 gaaggtcgga gtcaacggat tgaatcgagc ccacgacctt g 41 <210> 5 <211> 41 <212> DNA <213> Artificial Sequence <400> 5 gaaggtgacc aagttcatgc tgaatcgagc ccacgacctt c 41 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <400> 6 gtaatagtgc acggtagaat gacg 24

Claims

1. A primer set for detecting SNP molecular markers associated with Bombyx mori nuclear polyhedrosis virus resistance, characterized in that: The primer set includes primer sets for amplifying the Chr3-746 marker and the Chr27-5071 marker, respectively; the primer set for amplifying the Chr3-746 marker includes the primer sequences shown in SEQ.ID.NO.1-3, and the primer set for amplifying the Chr27-5071 marker includes the primer sequences shown in SEQ.ID.NO.4-6; the base polymorphism of the Chr3-746 marker is G / A; the base polymorphism of the Chr27-5071 marker is C / G.

2. The primer set according to claim 1, characterized in that In the primer set for amplifying the Chr3-746 marker, the primer shown in SEQ.ID.NO.1 is used to match the A allele, and the primer shown in SEQ.ID.NO.2 is used to match the G allele; in the primer set for amplifying the Chr27-5071 marker, the primer shown in SEQ.ID.NO.4 is used to match the G allele, and the primer shown in SEQ.ID.NO.5 is used to match the C allele.

3. The primer set according to claim 1 or 2, characterized in that The primer shown in SEQ.ID.NO.1 in the primer group for amplifying the Chr3-746 marker and the primer shown in SEQ.ID.NO.4 in the primer group for amplifying the Chr27-5071 marker are respectively labeled with HEX or VIC fluorescent probes; the primer shown in SEQ.ID.NO.2 in the primer group for amplifying the Chr3-746 marker and the primer shown in SEQ.ID.NO.5 in the primer group for amplifying the Chr27-5071 marker are respectively labeled with FAM fluorescent probes.

4. A product for detecting or assisting in detecting BmNPV resistance in silkworms, characterized in that: The product includes the primer set according to any one of claims 1 to 3; the product includes a detection reagent, a kit or a chip.

5. Use of the primer set according to any one of claims 1 to 3 or the product according to claim 4 in any of the following aspects: (1) Identification of silkworm varieties resistant to BmNPV; (2) Used for the purity identification of BmNPV-resistant silkworm varieties; (3) Identification of BmNPV disease resistance traits in silkworms; (4) Used for breeding or auxiliary breeding of silkworm strains resistant to BmNPV; (5) Localization and identification of BmNPV disease resistance genes in silkworms.

6. The use according to claim 5, characterized in that: The method comprises the following steps: extracting genomic DNA of a silkworm sample to be tested; detecting gene polymorphism or genotype of Chr3-746 marker and Chr27-5071 marker on the extracted genomic DNA; and making judgment, identification or assisting breeding according to the test results.

7. The application according to claim 6, wherein The method for judging, identifying or assisting breeding of the test results is as follows: if the genotype of the Chr3-746 marker is GG or the genotype of the Chr27-5071 marker is CC, the silkworm to be tested is a BmNPV-susceptible silkworm; if the genotype of the Chr3-746 marker is AA or AG and the genotype of the Chr27-5071 marker is GG or GC, the silkworm to be tested is a BmNPV-resistant silkworm.

8. The application according to claim 6, characterized in that, The KASP technology is used to detect gene polymorphism or genotype. The PCR amplification system of the KASP technology is calculated in 5 μL as follows: 2.5 μL KASP Master Mix (2×), 0.7 μL KASP primer set, 1 μL genomic DNA, and ddH2O were added to make up the system to 5 μL.

9. The application according to claim 8, characterized in that, The KASP primer set includes 0.15 μL each of F1 and F2 primers at a concentration of 10 μM, and 0.4 μL of R primer at a concentration of 10 μM; the genomic DNA concentration is 20-50 ng / μL.

10. The application according to claim 8, wherein The PCR amplification procedure of the KASP technology is as follows: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 20 s, annealing at 65°C for 1 min, with the annealing temperature decreasing by 0.8°C each cycle, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing at 57°C for 1 min, for a total of 27 to 30 cycles; extension at 25°C for 30 s, and collection of fluorescence signals.

Images