Method for exploring gene associated with body color trait of juvenile of antheraea pernyi

Abstract

The present application belongs to the technical field of biological genetic engineering, and specifically discloses a method for excavating a gene associated with the body color trait of a tussock moth larva, constructing a tussock moth body color trait analysis population, and counting the body color trait phenotype value of the tussock moth body color trait analysis population; based on the Super-GBS technology, SNP markers are developed and typed at the whole genome level for tussock moth materials of the tussock moth body color trait analysis population, and a genetic linkage map is drawn; the body color trait is positioned by QTL using the genetic linkage map, the corresponding linkage group physical position of the body color trait is obtained, and the body color trait GWAS analysis is simultaneously carried out; according to the gene information obtained from the two methods, the associated regions are compared with a database and annotated, and candidate genes of the body color trait of the tussock moth larva are excavated. The present application obtains relevant gene information by the two methods of QTL and GWAS, compares the information with a database and annotates the information, and excavates candidate genes of the body color trait of the tussock moth, so that the candidate genes of the body color trait of the tussock moth at the whole genome level are effectively and accurately realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological genetic engineering, and particularly to a method for excavating a gene associated with the body color trait of a tussah moth larva. BACKGROUND

[0002] The tussah moth (Antheraea pernyi) originated in China, and the tussah moth industry has a history of more than 2,000 years in China. The tussah moth germplasm resources in China account for more than 98% of the world's tussah moth germplasm resources. The tussah moth germplasm resources have been formed through thousands of years of natural evolution under different ecological conditions and contain various potential available genes, which are the precious biological resource wealth of the country. The excavation of the genes associated with important traits of the tussah moth germplasm resources lays a rich theoretical foundation for the origin and evolution of the tussah moth and the breeding of new varieties, and is also an inevitable way for the development of tussah moth scientific research. Therefore, the excavation of the candidate genes of important traits of the tussah moth germplasm resources has a promoting effect on the identification and innovative use of the germplasm resources and the development of tussah moth molecular marker-assisted breeding technology, and has a unique biological significance for the development of the tussah moth industry and the protection of the tussah moth germplasm resources worldwide.

[0003] The body color trait of the tussah moth larva is used as the primary selection marker in the identification of germplasm resources and for evaluating the purity and stability of the variety. At present, the research on the body color trait of the tussah moth larva mainly focuses on discovering the genetic rules and excavating the related genes through transcriptomics, and there is no research on excavating the candidate genes of the body color trait of the tussah moth larva by using genetic maps. The Super-GBS technology is derived from the GBS technology and inherits the advantages of the GBS technology, such as saving time and low cost, and can find SNP markers and genotyping in the whole genome, solve the problem of balancing between the number of markers and the sequencing depth for reducing cost, avoid the loss of a large amount of data, and cause low sequence coverage of the sites. Quantitative Trait Locus (QTL) is a method that anchors molecular markers and trait genes on chromosomes by means of genetic maps, analyzes the linkage relationship between the target traits and the markers, and screens important trait genes. This method provides a favorable reference for accurately discovering important trait-related genes. Although the body color trait of the tussah moth belongs to a quality trait, the QTL analysis can obtain the related genes. Genome-Wide Association Study (GWAS) has the advantages of being fast, efficient, and accurate in screening important traits, especially complex trait-related genes in the research of animals and plants, and can make up for the low efficiency of QTL positioning.

[0004] Therefore, if the related gene information can be obtained based on the advanced simplified genome technology and combined with two analysis methods, the candidate genes of the body color trait of the tussah caterpillar larvae can be explored through annotation by comparison with a database, so that the tussah germplasm resources can be evaluated more effectively and accurately, and the protection research of the tussah breed right can be further deepened.

[0005] Therefore, it is urgent to develop a method for exploring the related genes of the body color trait of the tussah caterpillar larvae based on the Super-GBS technology. SUMMARY

[0006] In order to solve the above technical problems, the application provides a method for exploring the related genes of the body color trait of the tussah caterpillar larvae.

[0007] In order to achieve the above purpose, the application is implemented according to the following technical scheme:

[0008] The method for exploring the related genes of the body color trait of the tussah caterpillar larvae comprises the following steps:

[0009] S1, constructing a tussah body color trait analysis population and counting the body color trait phenotype values of the tussah body color trait analysis population;

[0010] S2, developing and typing the SNP markers of the tussah materials of the tussah body color trait analysis population at the whole genome level based on the Super-GBS technology, and drawing a genetic linkage map;

[0011] S3, using the genetic linkage map to locate the QTL of the body color trait, obtaining the physical position of the linkage group corresponding to the body color trait, and simultaneously carrying out the GWAS analysis of the body color trait, obtaining the gene information of the related regions according to the two means, comparing with a database and annotating, and exploring the candidate genes of the body color trait of the tussah caterpillar larvae.

[0012] Further, the step S1 specifically comprises:

[0013] S11, using two tussahs with different body colors as the female parent and the male parent, configuring a cross combination, backcrossing the F1 generation with the female parent tussah to obtain a BC1M analysis population;

[0014] S12, counting the body color trait phenotype values of the tussah body color trait analysis population:

[0015] counting the body color of the 5th instar larvae at the mature stage, the body color of the 5th instar larvae at the mature stage of the F1 generation, investigating the gender at the pupa stage, and investigating the gender at the pupa stage.

[0016] Further, the step S2 specifically comprises:

[0017] S21, tussah sample collection and sample DNA extraction:

[0018] DNA extraction: Using animal genome DNA extraction kit, DNA was extracted from the tail of mature pupae of 113 BC1M populations, 3 F1 individuals and the tail of two parent moths, and the integrity, purity and concentration of the extracted DNA were detected. After detection, the DNA was stored at -80℃ for standby use;

[0019] S22, Super-GBS library construction and high-throughput sequencing:

[0020] 1) PstI-HF / MspI was used to cut the DNA, and T4 ligase was used to add adapters and barcodes to the ends of the cut fragments;

[0021] 2) The magnetic bead recovery system was used to recover 300-700 bp DNA fragments, and the recovered fragments were subjected to PCR amplification and determination of PCR product concentration;

[0022] 3) The mixed library was sequenced;

[0023] S23, data quality control:

[0024] Stacks and fastp software were used to filter the quality of the obtained Raw Reads, and the filtering standards were: (1) remove adapter sequences; (2) remove Reads with non-AGCT bases greater than or equal to 5; (3) remove bases with 5' and 3' quality values less than Q20; (4) remove Reads with length less than 75 bp; (5) remove low-quality Reads with number of bases with quality less than 15 exceeding 40% of the total number of bases in the Reads;

[0025] S24, SNP information site detection and genotyping:

[0026] 1) Using the existing chromosome genome CRA002120 as a reference sequence, BWA and GATK were used for data alignment and SNP detection; combined with VCFools for further filtering, the sites suitable for genotyping were screened;

[0027] 2) Based on the genotyping results of the parents, the markers between the parents were developed, the parent polymorphic sites suitable for mapping markers were screened, and the corresponding offspring genotypes were encoded;

[0028] S25, construction of genetic linkage map:

[0029] Using joinmap software for further filtering, linkage groups were divided, and the filtering conditions were: (1) remove individuals with abnormal base number abnorma1 exceeding 20%; (2) remove partially separated sites with chi-square p value less than 0.005; (3) remove sites with more than 20% missing; (4) remove sites with completely consistent similarity; i.e. a genetic map was constructed.

[0030] Further, the step S3 specifically comprises:

[0031] S31, QTL positioning:

[0032] By means of MapQTL6 software, the interval mapping method is used to estimate the LOD value in a single linkage group and genome range at the level of α=0.05 as a threshold to analyze the QTL of the trait;

[0033] S32, whole genome association analysis:

[0034] The MLM model of EMMAX and GAPIT software is used to perform the correlation analysis between the SNP markers at the whole genome level, and the correlation heat map, QQ plot and Manhattan plot are drawn;

[0035] S33, candidate gene mining:

[0036] The SNP site corresponding to the larger LOD value in the QTL analysis of the body color trait and the significant P value in the correlation analysis is associated with the related sequence according to the site information, is searched in the reference genome, the candidate gene sequence information is obtained, and the BlastN comparison annotation is performed on the NCBI website, and the candidate gene of the body color trait is screened.

[0037] Further, the LOD setting range in the step S31 is 2-15, the scanning step is 1cM, and the replacement test is repeated 1000 times.

[0038] Compared with the prior art, the present application uses the simplified genome sequencing technology to develop the SNP markers at the whole genome level on the tussah silkworm materials including parents, 3 F1 generations, 110 BC1M populations, constructs the high-density genetic map based on the body color trait, combines the QTL positioning based on the body color trait phenotype value to obtain the linkage group physical position corresponding to the body color trait, and performs the whole genome correlation analysis based on the linkage disequilibrium LD principle. The related gene information is obtained through the QTL and GWAS methods, is compared with the database and is annotated, the candidate gene of the body color trait of the tussah silkworm is mined, and the candidate gene of the body color trait of the tussah silkworm at the whole genome level is effectively and accurately realized. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is the QTL positioning result of the body color trait of the tussah silkworm.

[0040] Figure 2 It is the Manhattan plot of the GWAS analysis of the body color trait of the tussah silkworm. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with examples. The specific examples described herein are only used to explain the present application and do not limit the present application.

[0042] The following describes the discovery of the body color trait associated gene of tussah caterpillar larvae using the small white caterpillar (recessive homozygote (rryyggbb)) preserved by Liaoning Sericultural Science Institute as the female parent, the body back of the larvae being moon white and the body side being light sky blue; H04 (homozygous yellow caterpillar variety (__YYGG__)) as the male parent, with the following operations:

[0043] 1. Construction of tussah caterpillar larvae body color trait analysis population

[0044] The small white caterpillar (recessive homozygote (rryyggbb)) preserved by Liaoning Sericultural Science Institute was used as the female parent, the body back of the larvae being moon white and the body side being light sky blue, and H04 (homozygous yellow caterpillar variety (__YYGG__)) as the male parent. In 2021, the parents were self-crossed in spring and autumn, and were screened according to the body color characteristics of the larvae to ensure the purity of the parents. In 2022, the purified parents were used as materials to configure hybrid combinations, and the F1 generation was raised outdoors. Inconsistent individuals were eliminated, and in 2022, the F1 generation was backcrossed with the female parent small white caterpillar in the fall, and the offspring in the good area were selected as the BC1M analysis population and raised outdoors. The parents, F1 and BC1M were raised in the same net room, and the leaves were fed to prevent mixing between the moth areas, and the quality and yield were ensured.

[0045] 2. Statistics of tussah analysis population body color trait phenotype value

[0046] The body color traits of the parents were investigated when the larvae developed to the fifth instar mature period, and the related data were recorded. The larvae were reared in single areas to prevent mixing; the body color of the larvae in the good area was investigated in the fifth instar mature period of the F1 generation, and the larvae were reared individually; the sex of the F1 generation was investigated during the pupation period; the body color of the BC1M generation was investigated in the fifth instar mature period, and the larvae with the same body color were reared individually; the sex of the BC1M population was investigated during the pupation period. The specific phenotype statistics are shown in Table 1.

[0047]

[0048] 3. Tussah sample collection and sample DNA extraction

[0049] DNA extraction from 113 BC1M population, 3 F1 generation individuals and their parents' pupal abdomen, using UPure e Tissue A DNA Kit, and using Nanodrop 2000 (Thermo Scientific, USA) and 1.5% agarose gel electrophoresis to detect the integrity, purity and concentration of the extracted DNA. The qualified samples (concentration > 50 ng / μl; total DNA > 2 μg.; OD 260 / 280 = 1.8-2.0) were stored at -80℃ for standby use;

[0050] 4. Super-GBS library construction and high-throughput sequencing

[0051] The genome DNA of A. pernyi was digested by PstI-HF / MspI, and the fragments were added with adapters and barcodes at both ends using T4 ligase. The fragments of 300-700 bp were recovered using an improved magnetic bead recovery system by adjusting the volume ratio of magnetic bead solution to ligation product. The recovered fragments were amplified by PCR (95℃ 30s, [95℃ 30s, 62℃ 20s, 68℃ 15s] x 16 cycles, 68℃ 5min, 4℃ ∞); the concentration of PCR product was determined using Qubit (Thermo Fisher Scientific, USA), and the concentration was required to be greater than 5 ng / μl. The mixed library was sequenced on machine (Illumina Nova, PE150).

[0052] 5. The raw Raw Reads data obtained by sequencing was filtered for quality using Stacks and fastp software to obtain available Clean Reads data. The filtering criteria were: 1) remove adapter sequences; 2) remove Reads with N (non-AGCT) bases greater than or equal to 5; 3) remove 5' and 3' quality value low Q20 bases; 4) remove Reads with length less than 75 bp; 5) remove low-quality Reads with more than 40% of the total number of bases with quality less than 15.

[0053] 6. SNP information site detection and genotyping

[0054] (1) The genome of A. pernyi (CRA002120, https: / / bigd.big.ac.cn / gsa / ) For the reference sequence, the filtered data were aligned with the reference genome using BWA software, SNP site mutation detection and screening were performed using GATK software, and QD >= 2.0 was selected as the standard; and the obtained SNP sites were further filtered using VCFools, with the following filtering standards: 1) retaining sites with a read support depth of not less than 4; 2) deleting sites with a minimum allele frequency (MAF) of less than 0.01; and 3) retaining sites that can be typed in 80% of individuals, and screening SNP sites that can be used for subsequent typing.

[0055] (2) Based on the typing detection results of the parents, the markers between the parents were developed, the parent polymorphic sites of the marker type were screened, and the genotype of the corresponding offspring was encoded.

[0056] 7. Genetic linkage map construction

[0057] Based on the offspring typing results, linkage groups were further filtered and divided by using joinmap software, and a genetic map was constructed. The filtering standards were: 1) excluding sites with an abnormal base individual number abnormal exceeding 20%; 2) excluding sites with a chi-square p value less than 0.005; 3) excluding sites with a deletion exceeding 20%; and 4) excluding sites with completely consistent similarity.

[0058] 8. QTL positioning

[0059] By using MapQTL6 software, interval mapping method was used to estimate the LOD value in the range of a single linkage group and genome at the level of α = 0.05 as a threshold for QTL analysis of the trait, the LOD setting range was 2-15, the scanning step was 1 cM, and the permutation test was repeated 1000 times. The LOD value in the range of a single linkage group at the level of α = 0.05 was used as a threshold, that is, when a LOD peak value was greater than or equal to the threshold, it was considered that there was a QTL at the site (referring to Figure 1 ), and the QTL statistical results of the body color trait of the oak silkworm larvae were as shown in Table 2.

[0060] Table 2

[0061]

[0062] 9. Whole genome association analysis

[0063] EMMAX and GAPIT software MLM (Mixed Linear Model) model were used for correlation analysis between the phenotypic traits and SNP markers, and a correlation heat map, QQ plot and Manhattan plot were drawn (referring to Figure 2), and the significant loci of the partial body color traits of the Antheraea pernyi are shown in Table 3.

[0064] Table 3

[0065]

[0066]

[0067] 10. Candidate gene mining

[0068] The SNPs corresponding to the LOD values greater than 3 in the statistical QTL analysis of the body color traits and the P values less than 0.000001 in the whole genome association analysis are associated with the related sequences according to the locus information, the candidate gene sequences are obtained by searching in the reference genome of the Antheraea pernyi, and the BlastN comparison and annotation are performed on the NCBI website, and the related candidate genes are screened to obtain the candidate genes of the body color traits, that is, the candidate genes of the body color traits of the Antheraea pernyi, and part of the candidate genes of the body color traits of the Antheraea pernyi are shown in Table 4.

[0069] Table 4

[0070]

[0071] It should be noted that although only part of the candidate genes of the body color traits of the Antheraea pernyi are given above, according to the above method, the related gene information is obtained by the QTL and GWAS methods, and the database is compared and annotated, and all the candidate genes of the body color traits of the Antheraea pernyi can be mined, so that the candidate genes of the body color traits of the Antheraea pernyi at the whole genome level can be effectively and accurately realized.

[0072] The technical scheme of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. A method for discovering genes associated with body color traits in tussah silkworm larvae, characterized in that, Includes the following steps: S1. Construct a population for analyzing the body color traits of the tussah silkworm and statistically analyze the phenotypic values ​​of the body color traits of the tussah silkworm population; S2. Based on Super-GBS technology, SNP markers were developed and genotyped at the whole genome level for the tussah silkworm body color trait analysis population, and a genetic linkage map was drawn. S3. QTL mapping of body color traits was performed using genetic linkage maps to obtain the physical location of the corresponding linkage groups. At the same time, GWAS analysis of body color traits was carried out. Gene information of associated regions was obtained using the two methods, compared with the database and annotated to discover candidate genes for body color traits in tussah silkworm larvae.

2. The method for discovering genes associated with body color traits in tussah silkworm larvae according to claim 1, characterized in that, Step S1 specifically includes: S11. Using two different body color varieties of tussah silkworm as the female and male parents, hybridization combinations were configured, and the F1 generation was backcrossed with the female parent tussah silkworm to obtain the BC1M analysis population. S12. Statistical analysis of the body color phenotypic values ​​of the tussah silkworm population: The body color of the 5th instar larvae of both parents at maturity was statistically analyzed. The body color of the 5th instar larvae of the F1 generation at maturity in the superior area and the sex were investigated during the emergence moth stage. The body color of the 5th instar larvae of the BC1M generation at maturity and the sex were investigated during the pupal stage.

3. The method for discovering genes associated with body color traits in tussah silkworm larvae according to claim 2, characterized in that, Step S2 specifically includes: S21. Collection of silkworm samples and extraction of DNA from samples: Using an animal genomic DNA extraction kit, DNA was extracted from the tails of mature pupae (containing 113 BC1M populations), three F1 generation individuals, and the tails of two parental moths during their emergence stages. After extraction, the integrity, purity, and concentration of the DNA were tested, and the DNA was stored at -80℃ for later use. S22, Super-GBS library construction and high-throughput sequencing: 1) DNA was digested with PstI-HF / MspI, and the digested fragments were ligated at both ends with T4 enzyme to add adapters and barcodes; 2) Using a magnetic bead recovery system, recover 300-700 bp DNA fragments, perform PCR amplification on the recovered fragments, and determine the concentration of the PCR product; 3) Sequencing the mixed library; S23, Data Quality Control: The obtained RawReads were quality filtered using Stacks and FastP software. The filtering criteria were: (1) removing the connector sequence; (2) removing Reads with non-AGCT bases greater than or equal to 5; (3) removing bases with a quality value lower than Q20 at the 5' end and 3' end; (4) removing Reads with a length less than 75 bp; and (5) removing low-quality Reads with a quality value lower than 15 that account for more than 40% of the total number of bases in the Reads. S24, SNP information locus detection and genotyping: 1) Using the existing tussah silkworm chromosome genome CRA002120 as a reference sequence, BWA and GATK were used for data alignment and SNP detection; VCFools were used for further filtering to screen for loci that could be used for genotyping. 2) Based on the parental genotyping results, develop markers between parents, screen parental polymorphic sites that meet the mapping marker type, and encode the corresponding offspring genotypes; S25. Construction of genetic linkage maps: After further filtering using the joinmap software, linkage groups were divided. The filtering conditions were: (1) removing individuals with more than 20% abnormal bases; (2) removing segregating sites with a chi-square p value of less than 0.005; (3) removing sites with more than 20% deletion; and (4) removing sites with completely identical similarity. Thus, a genetic map was constructed.

4. The method for discovering genes associated with body color traits in tussah silkworm larvae according to claim 3, characterized in that, Step S3 specifically includes: S31, QTL positioning: Using MapQTL6 software, the interval plotting method was used to estimate the LOD value at the α=0.05 level within a single linkage group and the genome as a threshold for QTL analysis of traits; S32. Genome-wide association analysis: MLM models using EMMAX and GAPIT software were used to perform genome-wide association analysis between SNP markers, and correlation heatmaps, QQplots, and Manhattan plots were generated. S33, Candidate Gene Discovery: In the QTL analysis of body color traits, the SNP loci with larger LOD values ​​and significant P-values ​​in the association analysis were identified. Based on the locus information, relevant sequences were associated with them, and candidate gene sequence information was obtained by searching in the reference genome. The sequence information was then compared and annotated using BlastN on the NCBI website to screen out candidate genes for body color traits.

5. The method for discovering genes associated with body color traits in tussah silkworm larvae according to claim 4, characterized in that, In step S31, the LOD setting range is 2 to 15, the scan step size is 1 cm, and the permutation test is repeated 1000 times.

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