A hercynium x chromosome snp marker combination, a genetic sex identification method and application thereof

Abstract

The present application discloses a kind of black soldier fly X chromosome SNP marker combination, genetic sex identification method and application thereof, it is related to molecular marker, genotyping and insect sex identification technical field.The twenty sites shown in SEQ ID NO:1 to SEQ ID NO:20 consist of the black soldier fly X chromosome SNP marker combination in the present application.The genotype and sequencing depth information of twenty sites of the individual to be tested are obtained when identifying, the proportion H of effective marker heterozygous site and the normalized depth R relative to the autosomal control depth are calculated, H and R are compared with preset threshold respectively, according to the comparison result, determine the individual to be tested is male, female or to be rechecked.The identification coincidence rate of the present application is 100% by calibrating sample verification, in the experiment of fixed removal part site, using original determination threshold still obtains the identification result of all samples consistent.The present application has less marker, and the risk of misjudgment can be reduced by joint determination of two indexes, has good fault tolerance, and is suitable for black soldier fly genetic sex identification and male and female classification.

Description

Technical Field

[0001] This invention relates to the fields of molecular markers, genotyping, and insect sex identification, specifically to a black soldier fly X chromosome SNP marker combination, a genetic sex identification method, and its application. Background Technology

[0002] Black soldier flies have value in organic waste conversion, feed protein production, and resource utilization. Accurate differentiation between males and females is crucial in breeding, parent stock selection, population structure research, and large-scale reproduction. However, some developmental stages or incomplete samples lack reliable external morphological characteristics, and manual observation is easily affected by the age of the insects, sample integrity, and the operator's experience, making early, rapid, and accurate sex determination difficult.

[0003] Sequencing depth and heterozygosity based on large segments or entire chromosomes of sex chromosomes can reflect sex differences, but this requires processing a large number of sequences or windows, resulting in significant computational costs and hindering the development of standardized, small detection panels. Furthermore, single-site sequencing is susceptible to random sequencing errors, site deletions, or population variations, leading to insufficient stability and making it difficult to meet practical application requirements.

[0004] In genomic breeding analyses such as whole-genome resequencing, population genetic analysis, parental mating, and genome selection, sample sex is also an important grouping and quality control information. Missing or incorrect sample sex registration can affect the interpretation of sex chromosome-related variations, sequencing depth, heterozygosity, and population structure results, thus impacting the reliability of the analytical conclusions.

[0005] Most existing methods rely on a single indicator for gender determination. When this indicator is affected by factors such as sample quality, sequencing depth, or experimental operation, it is prone to misjudgment and cannot effectively identify and verify suspicious results. There is a lack of error tolerance mechanisms and verification guidelines for abnormal samples.

[0006] Therefore, how to accurately identify the genetic sex of black soldier flies using a small number of fixed X chromosome SNP markers is a technical problem that urgently needs to be solved in this field.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for identifying the genetic sex of black soldier fly X chromosome SNP marker combinations, and its application, in order to solve the problems mentioned in the background section.

[0009] A black soldier fly X chromosome SNP marker combination, wherein the SNP marker combination consists of the following twenty SNP markers: (1) C / A polymorphism at position 101 of the sequence shown in SEQ ID NO:1; (2) The G / A polymorphism at position 101 of the sequence shown in SEQ ID NO:2; (3) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:3; (4) The A / C polymorphism at position 101 of the sequence shown in SEQ ID NO:4; (5) T / A polymorphism at position 101 of the sequence shown in SEQ ID NO:5; (6) C / T polymorphism at position 101 of the sequence shown in SEQ ID NO:6; (7) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:7; (8) A / G polymorphism at position 101 of the sequence shown in SEQ ID NO:8; (9) T / G polymorphism at position 101 of the sequence shown in SEQ ID NO:9; (10) C / A polymorphism at position 101 of the sequence shown in SEQ ID NO:10; (11) G / A polymorphism at position 101 of the sequence shown in SEQ ID NO:11; (12) The A / C polymorphism at position 101 of the sequence shown in SEQ ID NO:12; (13) G / A polymorphism at position 101 of the sequence shown in SEQ ID NO:13; (14) C / G polymorphism at position 101 of the sequence shown in SEQ ID NO:14; (15) The A / C polymorphism at position 101 of the sequence shown in SEQ ID NO:15; (16) The A / G polymorphism at position 101 of the sequence shown in SEQ ID NO:16; (17) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:17; (18) C / T polymorphism at position 101 of the sequence shown in SEQ ID NO:18; (19) A / G polymorphism at position 101 of the sequence shown in SEQ ID NO:19; (20) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:20.

[0010] Preferably, the physical positions of the twenty SNP markers on the X chromosome of the black soldier fly are as follows: positions 1,117,211, 4,871,650, 15,996,348, 17,916,890, 9,240,667, 7,986,203, 16,252,060, 5,741,374, and 15,853,7... 48th, 7,796,775th, 5,337,488th, 16,967,598th, 18,463,444th, 19,519,738th, 8,730,694th, 2,113,068th, 4,102,635th, 18,883,857th, 4,817,218th, 5,268,683rd.

[0011] Application of a black soldier fly X chromosome SNP marker combination as described above in the genetic sex determination of black soldier flies.

[0012] A method for genetic sex determination in black soldier flies, comprising the following steps: (1) Obtain the genotype and sequencing depth information of the twenty SNP markers mentioned above in the genome of the black soldier fly individual to be tested; (2) Calculate the proportion H of heterozygous sites of effective SNP markers and the normalized depth R relative to the depth of autosomal control in the same sample; (3) Compare H and R with preset thresholds respectively, and determine the genetic sex of the individual to be tested as male, female or to be retested based on the comparison results.

[0013] Preferably, the effective SNP marker in step (2) is defined as: an SNP marker with a complete diploid genotype, a sequencing depth DP≥5 and a genotype quality GQ≥20.

[0014] Preferably, the proportion H of heterozygous sites in step (2) is calculated according to the following formula: H = Nhet / K; Where K is the total number of valid SNP markers, and Nhet is the number of heterozygous genotype markers among the valid SNP markers.

[0015] Preferably, the normalized depth R in step (2) is calculated according to the following formula: R = (1 / K) × Σ(DPs / DA); Where K is the total number of valid SNP markers, DPs is the sequencing depth of the s-th valid SNP marker, and DA is the autosomal control depth of the same sample.

[0016] Preferably, in step (3), the preset H threshold is 0.10 and the preset R threshold is 0.9655591; When H < 0.10 and R < 0.9655591, it is determined to be male; When H ≥ 0.10 and R ≥ 0.9655591, it is determined to be female; When H < 0.10 and R ≥ 0.9655591, or H ≥ 0.10 and R < 0.9655591, it is determined to be pending re-inspection.

[0017] Preferably, when the total number of valid SNP markers K < 14, it is directly determined to be pending re-examination.

[0018] Preferably, the genotype and sequencing depth information in step (1) is obtained by any one of whole genome sequencing, target region capture sequencing or multiplex amplicon sequencing.

[0019] The present invention provides a black soldier fly X chromosome SNP marker combination, a genetic sex identification method, and its application, which have the following beneficial effects: (1) The present invention limits the sex determination object to 20 fixed SNP markers. Compared with the analysis method based on large segments or whole chromosomes of sex chromosomes, it greatly reduces the number of markers to be processed and the amount of calculation, which is convenient for developing standardized small detection panels.

[0020] (2) This invention uses two independent signals, heterozygosity and normalized depth, to determine gender. When the two indicators are in the same direction, the gender result is output. When the directions are not in the same direction, the result is output to be re-examined. This can effectively reduce the risk of misjudgment caused by interference from factors such as sample quality, sequencing depth or experimental operation of a single indicator. At the same time, it provides clear re-examination guidance for abnormal samples.

[0021] (3) The 20 loci set by this invention all obtained complete genotypes in the calibration samples, and the effective locus detection rate was high. When some markers failed to be detected, the remaining effective markers could still be used to complete the determination, which has good fault tolerance performance and improves the stability and applicability of the scheme in practical applications.

[0022] (4) The internal identification concordance rate of the 20-site scheme was 100% after verification with 23 male and 14 female calibration samples. In the experiments with fixed removal of 2, 4 or 6 sites, the same identification results were obtained for 37 samples using the original decision threshold without change, which further confirmed the robustness of the scheme. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the process for genetic sex determination in black soldier flies based on 20 X chromosome SNP markers.

[0024] Figure 2Scatter plot of the H-normalized depth R distribution of heterozygous sites in 37 black soldier fly samples. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] I. Design Concept of the Invention The twenty SNP markers used in this invention are shown in Table 1. All coordinates are 1-based and located on the black soldier fly X chromosome sequence used in this application; each point is also defined by the 201 bp flanking sequences shown in SEQ ID NO:1-20.

[0027] Table 1. Twenty Fixed SNP Markers

[0028] The flanking sequences shown in SEQ ID NO:1-20 were extracted from the black soldier fly reference genome sequence used during marker screening. Each sequence consists of 100 bp upstream of the SNP, the reference allele at position 101, and 100 bp downstream, for a total length of 201 bp; the 101st base has been matched line by line with the REF allele in the combined typing VCF.

[0029] The genotype, sequencing depth (DP), and genotype quality (GQ) of the test samples were detected at twenty loci shown in Table 1. In this embodiment, loci with a complete diploid genotype, DP ≥ 5, and GQ ≥ 20 were defined as valid loci.

[0030] Let K be the number of valid loci and Nhet be the number of heterozygous genotype loci, then the heterozygosity index H is: H = Nhet / K; For example, if all twenty markers in a sample to be tested pass quality control, then K=20; if one marker is a heterozygous genotype and the other nineteen are homozygous genotypes, then Nhet=1, and the H of this sample is 1 / 20=0.05.

[0031] Let the sequencing depth of the s-th valid SNP marker be DPs, and the sequencing depth of the autosomal control in the same sample be DA, then the normalized depth index R is: R = (1 / K) × Σ(DPs / DA); For example, still taking K=20, if the average sequencing depth DA of all qualified SNP loci on chromosome 6 of this sample is 30, and the total DP of the twenty target markers is 360, then Σ(DPs / DA)=360 / 30=12, R=(1 / 20)×12=0.60. In this case, H=0.05 and R=0.60, both indicators are below the threshold of this embodiment, and the sample is determined to be male. As another example, if another sample to be tested has K=20, Nhet=3, DA=30, and the total DP of the twenty target markers is 660, then H=3 / 20=0.15, R=(1 / 20)×(660 / 30)=1.10, both indicators are not below the threshold of this embodiment, and the sample is determined to be female. The above values ​​are only used to illustrate the calculation process.

[0032] In this embodiment, DA is calculated using the average depth (DP) of all eligible SNP loci on chromosome 6 from the same sample. The control SNPs are biallelic SNPs with a filter of PASS or ".", a QUAL ≥ 30, and loci participating in the DA calculation for a single sample have a complete diploid genotype, DP ≥ 3, and GQ ≥ 20; the locus deletion rate does not exceed 0.10, and the minor allele count (MAC) ≥ 2. Other embodiments may use the median depth of all eligible loci on chromosome 6, or the average or median depth of one or more uniquely comparable regions on autosomes; when changing the depth control, the R threshold should be recalibrated using samples of known sex.

[0033] When using the criteria calibrated with this data: H < 0.10 and R < 0.9655591 indicates male; H ≥ 0.10 and R ≥ 0.9655591 indicates female; when H < 0.10 and R ≥ 0.9655591, heterozygosity supports male and depth supports female; when H ≥ 0.10 and R < 0.9655591, heterozygosity supports female and depth supports male. Both of these situations indicate inconsistency between the two indicators, and a retest should be initiated instead of a mandatory sex determination. Inconsistency may be caused by insufficient sequencing depth, sample mixing or contamination, abnormal alignment, genotyping errors at individual loci, or abnormal chromosomal composition. Additionally, a retest should be initiated if there are fewer than fourteen valid loci or other quality control failures. When the detection platform, library method, or depth control method changes, a known sex calibration sample can be used to redetermine the threshold.

[0034] II. Specific Application Cases Example 1: Sex determination of 37 samples using 20 SNP markers 1. Samples and candidate sites After the combined genotyping data underwent quality control to ensure consistency between the sample labels and the overall X chromosome signal, twenty-three male samples and fourteen female samples were selected as the thirty-seven calibration samples for this embodiment. Candidate loci were required to be biallelic SNPs in the above samples, with a filter score of PASS or ".", a QUAL ≥ 30, and each sample must have a complete diploid genotype, DP ≥ 5, and GQ ≥ 20.

[0035] 2. Tag Combinations and Calculations Twenty loci were selected from the candidate loci as shown in Table 1. For each sample, the H and R values ​​for the twenty loci were calculated, and a threshold was set at the midpoint between the maximum value in the male group and the minimum value in the female group. The resulting heterozygosity threshold TH was 0.10, and the normalized depth threshold TR was 0.9655591.

[0036] 3. Results The range of H for males was 0.000000–0.050000, and for females it was 0.150000–0.500000; the range of R for males was 0.621964–0.785069, and for females it was 1.146049–1.538794. There was no overlap between the male and female ranges for either indicator. All twenty-three male samples were identified as male, and all fourteen female samples were identified as female. There were 37 correct identifications, 0 pending retesting, and 0 incorrect identifications. The internal identification accuracy rate for this embodiment was 37 / 37 × 100% = 100%. Results for each sample are shown in Table 2, and the distribution is shown in […]. Figure 2 .

[0037] Table 2. Identification results of 20 SNP markers in 37 samples.

[0038] Example 2: Identification effect after partial removal of markers 1. Experimental Design To evaluate the fault tolerance when effective genotypes were not obtained at some loci, the same thirty-seven samples as in Example 1 and fixed decision thresholds TH=0.10 and TR=0.9655591 were used to set four scenarios: 20 complete loci, 18 loci retained after removing SEQ ID NO:19-20, 16 loci retained after removing SEQ ID NO:17-20, and 14 loci retained after removing SEQ ID NO:15-20. In each scenario, only the retained loci were used to recalculate H and R; no retraining or threshold adjustment was performed.

[0039] 2. Results Table 3. Identification results after partial removal of SNP markers.

[0040] The results showed that, under the fixed deletion order, even after removing six sites and retaining only SEQ ID NO:1-14, all thirty-seven samples still yielded results consistent with the registered sex: 37 correct, 0 requiring retesting, and 0 incorrect, with an internal identification concordance rate of 100%. This example demonstrates that even when individual marker detection fails, calculating H and R using the remaining valid markers can still maintain the determination. These results pertain to the fixed deletion combinations listed in Table 3 and should not be interpreted as implying that any subset of fourteen sites exhibits the same performance.

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A black soldier fly X chromosome SNP marker combination, characterized in that, The SNP tag combination consists of the following twenty SNP tags: (1) C / A polymorphism at position 101 of the sequence shown in SEQ ID NO:1; (2) The G / A polymorphism at position 101 of the sequence shown in SEQ ID NO:2; (3) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:3; (4) The A / C polymorphism at position 101 of the sequence shown in SEQ ID NO:4; (5) T / A polymorphism at position 101 of the sequence shown in SEQ ID NO:5; (6) C / T polymorphism at position 101 of the sequence shown in SEQ ID NO:6; (7) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:7; (8) A / G polymorphism at position 101 of the sequence shown in SEQ ID NO:8; (9) T / G polymorphism at position 101 of the sequence shown in SEQ ID NO:9; (10) C / A polymorphism at position 101 of the sequence shown in SEQ ID NO:10; (11) G / A polymorphism at position 101 of the sequence shown in SEQ ID NO:11; (12) The A / C polymorphism at position 101 of the sequence shown in SEQ ID NO:12; (13) G / A polymorphism at position 101 of the sequence shown in SEQ ID NO:13; (14) C / G polymorphism at position 101 of the sequence shown in SEQ ID NO:14; (15) The A / C polymorphism at position 101 of the sequence shown in SEQ ID NO:15; (16) The A / G polymorphism at position 101 of the sequence shown in SEQ ID NO:16; (17) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:17; (18) C / T polymorphism at position 101 of the sequence shown in SEQ ID NO:18; (19) A / G polymorphism at position 101 of the sequence shown in SEQ ID NO:19; (20) T / C polymorphism at position 101 of the sequence shown in SEQ ID NO:

20.

2. The black soldier fly X chromosome SNP marker combination according to claim 1, characterized in that, The physical locations of the twenty SNP markers on the X chromosome of the black soldier fly are, in order: positions 1,117,211, 4,871,650, 15,996,348, 17,916,890, 9,240,667, 7,986,203, 16,252,060, 5,741,374, and 15,853,748. The positions are: 7,796,775, 5,337,488, 16,967,598, 18,463,444, 19,519,738, 8,730,694, 2,113,068, 4,102,635, 18,883,857, 4,817,218, and 5,268,683.

3. The application of the black soldier fly X chromosome SNP marker combination as described in claim 1 in the genetic sex identification of black soldier flies.

4. A method for genetic sex determination in black soldier flies, characterized in that, Includes the following steps: (1) Obtain the genotype and sequencing depth information of the twenty SNP markers described in claim 1 in the genome of the black soldier fly individual to be tested; (2) Calculate the proportion of heterozygous sites H of effective SNP markers, and the normalized depth R relative to the depth of autosomal control in the same sample; (3) Compare H and R with preset thresholds respectively, and determine the genetic sex of the individual to be tested as male, female or to be retested based on the comparison results.

5. The method for genetic sex determination of black soldier fly larvae according to claim 4, characterized in that, The effective SNP markers mentioned in step (2) are defined as: SNP markers with complete diploid genotype, sequencing depth DP≥5 and genotype quality GQ≥20.

6. The method for genetic sex determination of black soldier fly larvae according to claim 4, characterized in that, The proportion H of heterozygous sites mentioned in step (2) is calculated according to the following formula: H = Nhet / K; Where K is the total number of valid SNP markers, and Nhet is the number of heterozygous genotype markers among the valid SNP markers.

7. The method for genetic sex determination of black soldier fly larvae according to claim 4, characterized in that, The normalized depth R mentioned in step (2) is calculated according to the following formula: R = (1 / K) × Σ(DPs / DA); Where K is the total number of valid SNP markers, DPs is the sequencing depth of the s-th valid SNP marker, and DA is the autosomal control depth of the same sample.

8. The method for genetic sex determination of black soldier fly larvae according to claim 4, characterized in that, In step (3), the preset H threshold is 0.10 and the preset R threshold is 0.9655591; When H < 0.10 and R < 0.9655591, it is determined to be male; When H ≥ 0.10 and R ≥ 0.9655591, it is determined to be female; When H < 0.10 and R ≥ 0.9655591, or H ≥ 0.10 and R < 0.9655591, it is determined to be pending re-inspection.

9. The method for genetic sex determination of black soldier fly larvae according to claim 4, characterized in that, When the total number of valid SNP markers K < 14, it is directly determined to be pending re-examination.

10. The method for genetic sex determination of black soldier fly larvae according to claim 4, characterized in that, The genotype and sequencing depth information mentioned in step (1) is obtained by any one of the following methods: whole genome sequencing, target region capture sequencing, or multiplex amplicon sequencing.

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