Method for identifying gender of poultry based on high-throughput sequencing

Abstract

The invention provides a method for identifying the gender of poultry based on high-throughput sequencing, which is based on a sequencing principle, adopts any method including whole genome re-sequencing, targeted genome sequencing, low-depth re-sequencing, first-generation sequencing and the like to obtain the sequencing depth of an autosome and a Z chromosome, and judges the individual gender according to the ratio of the autosome to the Z chromosome. The method does not depend on molecular markers, the off-target problem does not exist, variation sites obtained through sequencing can be used for other biological information analysis, and the method has great significance in poultry breeding technology development.

Description

Technical Field

[0001] The invention relates to the technical field of poultry sex identification, and in particular to a method for identifying the sex of poultry based on high-throughput sequencing. Background Art

[0002] In livestock and poultry breeding, it is of great significance to identify the sex of livestock and poultry as early as possible. It can be grouped according to male and female as early as possible. Not only can it be grouped according to different breeding directions, but also appropriate feeding methods can be formulated for different genders to improve production efficiency. It is difficult to distinguish the sex of newly hatched chicks from the appearance. Only when they grow to two or three months old can the male and female be distinguished by their fur color and comb. At present, the schemes for sex identification of poultry (mainly chickens and ducks) are mainly divided into three categories: the first category is anal inversion identification, the second category is identification based on fast and slow feathers, and the third category is identification using molecular means.

[0003] Anal inversion identification refers to determining the sex of an individual bird by examining the internal structure of the cloaca (chicks) or the genital protrusion (ducklings). This identification method is suitable for chicks within a few hours after hatching, otherwise the anus of the chicks will be tight and difficult to open, making it inconvenient to observe. In addition, this method is also more dependent on the professionalism of the staff and can cause significant and irreversible damage to the chicks.

[0004] Fast and slow feather identification is to judge the sex of an individual by observing the length of the primary wing feathers and the covert wing feathers. Specifically, the primary wing feathers that are longer than the covert wing feathers are fast feathers, and the primary wing feathers that are shorter than the covert wing feathers are slow feathers. The principle of this identification method is that the gene that controls the growth rate of feathers exists on the sex chromosomes. If a fast-feathered rooster is crossed with a slow-feathered hen, the offspring will all have female fast feathers and male slow feathers. However, this identification method is only applicable to specific specialized strains.

[0005] In large-scale industrial farming, molecular identification is generally preferred, mainly using molecular markers for sex identification. Based on the differences in sex chromosomes, especially the sex-specific genes on the W chromosome (such as the CHD gene of chickens), PCR technology can be used to amplify the sex-specific genes and then perform first-generation sequencing for sex identification. This identification method has high accuracy, but is costly. Molecular markers may differ between varieties, and some varieties require a large amount of sequencing data to locate molecular markers for sex identification. The process of locating molecular markers requires a certain bioinformatics foundation. The implementation of the solution is complex and costly.

[0006] In addition, it can also be identified through genotyping, which generally uses first-generation sequencing. The typing accuracy is relatively high but the throughput is relatively low. The number of molecular markers obtained is limited. The typing results cannot be used for other bioinformatics analysis except gender identification, such as whole genome association analysis, whole genome selection, etc. Whole genome resequencing can capture all variations in the genome, but sequencing is random and there is a risk of not capturing the target marker. In addition, the polymorphism of this type of molecular marker is low and not easy to be found. At present, the cost of resequencing is high, and there are certain difficulties in large-scale application in breeding. Summary of the invention

[0007] The present invention provides a method for identifying the sex of poultry based on high-throughput sequencing, which is used to solve the defects of relying on molecular markers and having off-target risks in the existing technology of identifying the sex of poultry by genetic means. The method is based on the sequencing principle (whole genome resequencing, low-depth resequencing and first-generation sequencing can be realized), calculates the sequencing depth of autosomes and Z chromosomes, and calculates the ratio RATE of the average sequencing depth of autosomes and Z chromosomes. The sex of an individual is determined according to the ratio RATE, so as to realize convenient, fast, high-throughput and molecular marker-independent identification of the sex of poultry.

[0008] In a first aspect, the present invention provides a method for sex identification of poultry, which obtains the sequencing depth of the target autosomes and Z chromosomes through gene sequencing, and the ratio of the autosome to the Z chromosome sequencing depth RATE normal distribution mean is close to 1 for males, and the RATE normal distribution mean is close to 2 for females.

[0009] Poultry such as chickens, ducks, and geese are diploid, with autosomes in pairs and sex chromosomes of ZZ (male) or ZW (female). The sequencing depth actually reflects the abundance of the corresponding chromosome in the genome, so the RATE value is 1 or 2. Since sequencing has a certain degree of randomness, the ratio is not strictly 1 or 2, but a normal distribution with a mean of 1 or 2. Since there are two autosomes, if RATE is close to 1, it can be determined that there are also two Z chromosomes, and the sample is of ZZ genotype, that is, male; if RATE is close to 2, it can be determined that there is only one Z chromosome, and the sample is of ZW genotype, that is, female.

[0010] Preferably, the gene sequencing described in the above method includes but is not limited to whole genome resequencing, low-depth resequencing and first-generation sequencing.

[0011] Preferably, the above method comprises the following steps: (1) Extract DNA from the individual to be tested; (2) Construction of DNA library; (3) DNA library is sequenced to obtain fastq files; (4) Analyze the fastq file data to obtain the VCF file; (5) Obtain the average sequencing depth of autosomes and chromosomes Z from the VCF file, and calculate the ratio of the average sequencing depth of autosomes to chromosomes Z, RATE; (6) If the mean of the RATE normal distribution is close to 1, the individual to be tested is male; if the mean of the RATE normal distribution is close to 2, the individual to be tested is female.

[0012] Since sequencing has a certain randomness, the captured reads (sequencing depth) also have a certain randomness. Therefore, the RATE value is normally distributed with a mean of 1 or 2. In some special cases, there may be certain deviations. For example, there may be a normal distribution with a mean of 0.8 and 1.6. At this time, the clustering method can be used to divide all individuals into two categories, the one with a low mean is ZZ, which is male, and the one with a high mean is ZW, which is female.

[0013] Further preferably, the two primer sequences used for constructing the DNA library in the above step (2) are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0014] Further preferably, the method for obtaining the average sequencing depth of the autosomes and the Z chromosome in the above step (5) is: the method for obtaining RATE is specifically to extract the DP field in the INFO information of each site in the VCF file as the sequencing depth of the site; extract the sequencing depth of all sites on all autosomes as the sequencing depth of the autosomes; extract the sequencing depth of all sites on all Z chromosomes as the sequencing depth of the Z chromosome. The chromosome information can be obtained from the chrom field of the VCF file.

[0015] More preferably, the data analysis described in step (4) above further includes the following steps: (a) Off-machine data quality control: fastp software version 0.20.1 was used to quality control and filter fastq files into clean fastq files, with the minimum quality threshold set to 20 and the minimum average quality threshold set to 40; (b) Reference genome alignment: Use sentieon software version 202010.02 and samtools software version 1.3 to align the clean fastq file with the reference genome to generate a bam file; (c) BAM file sorting: Use sentieon software with version number 202010.02 and samtools software with version number 1.3 to sort the BAM files; (d) Mutation detection: Use the HaplotypeCaller method of GATK4 to perform mutation detection and generate a VCF file.

[0016] More preferably, the reference genome in step (b) is determined according to the poultry species being tested, and its source can be any complete genome of the poultry species on NCBI.

[0017] Preferably, the poultry in the above method is poultry with male sex chromosomes being ZZ and female sex chromosomes being ZW, including chickens, ducks and geese.

[0018] In a second aspect, the present invention provides application of the above method in livestock sex identification.

[0019] The present invention essentially utilizes the different gene abundances of sex chromosomes and autosomes of female and male animals to identify their sex. This principle can also be used for livestock or aquatic products such as pigs, cattle, and sheep. Taking livestock as an example, their sex chromosomes XY are male and XX are female. The average sequencing depth of their autosomes and X chromosomes can be detected and their ratio can be calculated. Since there are two autosomes, if RATE is close to 1, it can be determined that there are also two X chromosomes, and the sample is XX genotype, that is, female; if RATE is close to 2, it can be determined that there is only one X chromosome, and the sample is XY genotype, that is, male.

[0020] In a third aspect, the present invention provides the application of the variant sites captured by the above method in bioinformatics analysis.

[0021] The method for sex identification of poultry provided by the present invention is based on the sequencing principle (whole genome resequencing, low-depth resequencing and first-generation sequencing can all be achieved), calculates the sequencing depth of autosomes and chromosomes Z, and obtains the ratio RATE of the average sequencing depth of autosomes and chromosomes Z. The sex of an individual is determined based on the ratio RATE. This method does not rely on molecular markers, does not have the risk of off-target, and can conveniently and quickly identify the sex of poultry with high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 It is a visualization diagram of the high-throughput results obtained in Example 5 of the present invention.

[0024] Figure 2 It is a schematic diagram of the high-throughput result file obtained in Example 5 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1 DNA extraction of test samples The kit used for DNA extraction in this example is GenoPrep® Blood DNA Rapid Extraction Kit (Magnetic Bead Method) produced by Shijiazhuang Boredi Biotechnology Co., Ltd., item number: DN004006, and the sample is 10 μl of duck blood.

[0027] Sample DNA was extracted according to the kit instructions and the DNA concentration was quantified using Qubit® 2.0.

[0028] Example 2 DNA library construction The kit used for DNA library construction in this example is GenoBaits® DNA Library Prep Kit for MGI V4.0 from Shijiazhuang Boredy Biotechnology Co., Ltd., catalog number: GB000204. The specific steps are as follows: Step 1: DNA fragmentation and end repair plus A (30~50min) 1. Prepare the end repair reaction system according to the reagents and amounts shown in Table 1.

[0029] 2. Vortex the reaction system to mix well and centrifuge briefly.

[0030] 3. Place the PCR tube / plate containing the reaction system in the PCR instrument, set the PCR amplification program according to Table 2, and set the hot cover temperature to 82°C.

[0031] 4. When the temperature reaches 4°C, proceed to step 2: connector connection.

[0032] Step 2: Connector connection (70 minutes) 1. According to the reagents and amounts shown in Table 3, directly add the connector connection system into the system in step 1.3.

[0033] 2. Vortex the reaction system to mix well and centrifuge briefly.

[0034] 3. Place the PCR tube / plate containing the reaction system in the PCR instrument, set the PCR amplification program according to Table 4, and remove the hot cover.

[0035] 4. When the temperature reaches 4°C, proceed to step 3: connector connection system purification.

[0036] Step 3: Connector ligation system purification (30-65min) 1. Add 54 μL GenoPrep® DNA Clean Beads to the ligation system in step 2.3, vortex to mix well, avoid generating bubbles during vortexing, let stand at room temperature for 5 min, and then centrifuge briefly.

[0037] 2. Place the PCR tube / plate on the magnetic rack and let it stand at room temperature for at least 3 min until the solution is clear. Carefully remove the supernatant (retain the magnetic beads) making sure not to absorb the magnetic beads.

[0038] 3. Keep the PCR tube / plate on the magnetic rack, add 100 μL 80% ethanol, let it stand at room temperature for 30 seconds, carefully remove all the supernatant (keep the magnetic beads), and let it stand at room temperature for at least 5 minutes until the ethanol is completely evaporated.

[0039] 4. Remove the PCR tube / plate from the magnetic rack and immediately resuspend the magnetic beads using the PCR system in step 4.1.

[0040] Step 4: Library amplification (30 min) 1. In step 3.4, prepare the PCR amplification system in the PCR tube / plate according to Table 5, wherein the MGII5 sequence is shown as SEQ ID NO.1, and the IDXI7 sequence is shown as SEQ ID NO.2.

[0041] 2. Vortex the reaction system to mix well and centrifuge briefly.

[0042] 3. Place the PCR tube / plate containing the reaction system into the PCR instrument and set the PCR amplification program according to Table 6. The heated cover should be set to 105°C.

[0043] 4. Select the appropriate number of cycles according to Table 7 5. When the above procedure reaches 4°C, the PCR amplification system can be stored at -20°C overnight.

[0044] Step 5: Purification (40 min) 1. Add 40 μL GenoPrep® DNA Clean Beads to the system in step 4.3 and vortex to mix. Do not generate bubbles during vortexing. After standing at room temperature for 5 minutes, briefly centrifuge.

[0045] 2. Place the PCR tube / plate on the magnetic rack and let it stand at room temperature for at least 3 minutes until the solution is clear. Carefully remove the supernatant (retain the magnetic beads), making sure not to absorb the magnetic beads.

[0046] 3. Keep the PCR tube / plate on the magnetic rack, add 100μL 80% ethanol, and let it stand at room temperature for 30 seconds. Carefully remove the supernatant (keep the magnetic beads) and let it stand at room temperature for 5 minutes until the ethanol is completely evaporated.

[0047] 4. Remove the PCR tube / plate from the magnetic rack, add 45 μL 10 mM Tris-HCl, vortex to mix, let stand at room temperature for 5 minutes, and then centrifuge briefly.

[0048] 5. Place the PCR tube / plate on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution becomes clear.

[0049] 6. Transfer the supernatant to a new 0.2 mL low-adsorption PCR tube / plate (retain the supernatant), making sure not to absorb the magnetic beads.

[0050] 7. The purified DNA library was stored at -20°C.

[0051] Example 3 DNA library sequencing The sequencing was performed using the DNBSEQ-T7RS high-throughput sequencer of Shenzhen MGI Technology Co., Ltd., with its dedicated DNBSEQ-T7RS (FCL PE100) V3.0 reagent set, item number: 940-000269-00. DNB (DNA Nanoball) was prepared and the sample was loaded according to the kit instructions, and sequencing was started according to the instrument manual to obtain the fastq file as the result.

[0052] Example 4 Off-machine data processing This embodiment is a method for processing a fastq file obtained by sequencing, which comprises the following steps: 1. Data quality control: Use fastp (version 0.20.1) to perform quality control and filtering on the fastq files, with the minimum quality threshold set to 20 and the minimum average quality threshold set to 40; generate clean fastq files.

[0053] 2. Reference genome alignment: Use sentieon (version 202010.02) and samtools (version1.3) to align the quality-controlled clean fastq with the target reference genome CAU_duck1.0 (GCA_002743455.1), which can be directly obtained from the NCBI website. The generated bam file is used for the subsequent variant detection process.

[0054] 3. Sorting bam files: Use sentieon (version 202010.02) and samtools (version1.3) tools to sort bam files.

[0055] 4. Mutation detection: Use the HaplotypeCaller method of GATK4 to perform mutation detection and generate a VCF file.

[0056] Example 5 Obtaining Results This embodiment is a method for obtaining a result according to a VCF file, which comprises the following steps: 1. Extract the DP field in the INFO information of each site in the VCF file as the sequencing depth of the site; extract the sequencing depth of all sites on all autosomes as the sequencing depth of the autosomes; extract the sequencing depth of all sites on all Z chromosomes as the sequencing depth of the Z chromosome. The above content can be completed with the assistance of a computer.

[0057] 2. Calculate the ratio of the average sequencing depth of the autosomes to the average sequencing depth of the Z chromosome. RATE will be presented as two normal distributions with means of 1 and 2 respectively. When the RATE normal distribution is close to 1, it is male, and when the RATE normal distribution is close to 2, it is female. The single result is as follows: Figure 1 shown.

[0058] The present application performed sex identification on 1750 adult ducks as in Examples 1-5, and the high-throughput results are as follows Figure 2 As shown, after comparing with the results obtained by direct observation of gender, it was found that the accuracy of the gender identification results obtained by the above method of the present application can reach 100%.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying the sex of poultry based on high-throughput sequencing, characterized in that: The sequencing depth of the target autosomes and Z chromosomes is obtained through gene sequencing. The ratio of the sequencing depth of the autosomes to the Z chromosome RATE has a normal distribution mean close to 1 for males, and a normal distribution mean close to 2 for females.

2. The method according to claim 1, characterized in that The gene sequencing includes but is not limited to first generation sequencing technology, second generation sequencing technology and third generation sequencing technology.

3. The method according to any one of claims 1 or 2, characterized in that: The method comprises the following steps: (1) Extract DNA from the individual to be tested; (2) Construction of DNA library; (3) DNA library is sequenced to obtain fastq files; (4) Analyze the fastq file data to obtain the VCF file; (5) Obtain the average sequencing depth of autosomes and chromosomes Z from the VCF file, and calculate the ratio of the average sequencing depth of autosomes to chromosomes Z, RATE; (6) If the mean of the RATE normal distribution is close to 1, the individual to be tested is male; if the mean of the RATE normal distribution is close to 2, the individual to be tested is female.

4. The method according to any one of claims 1 to 3, characterized in that: The sequences of the two primers used in the construction of the DNA library in step (2) are shown as SEQ ID NO.1 and SEQ ID NO.

2.

5. The method according to any one of claims 1 to 3, characterized in that: The method for obtaining the average sequencing depth of the autosomes and the Z chromosome in step (5) is as follows: extract the DP field in the INFO information of each site in the VCF file as the sequencing depth of the site; extract the sequencing depth of all sites on all autosomes as the sequencing depth of the autosomes; extract the sequencing depth of all sites on all Z chromosomes as the sequencing depth of the Z chromosome.

6. The method according to any one of claims 1 to 3, characterized in that: The data analysis described in step (4) also includes the following steps: (a) Off-machine data quality control: fastp software version 0.20.1 was used to quality control and filter fastq files into clean fastq files, with the minimum quality threshold set to 20 and the minimum average quality threshold set to 40; (b) Reference genome alignment: Use sentieon software version 202010.02 and samtools software version 1.3 to align the clean fastq file with the reference genome to generate a bam file; (c) BAM file sorting: Use sentieon software with version number 202010.02 and samtools software with version number 1.3 to sort the BAM files; (d) Mutation detection: Use the HaplotypeCaller method of GATK4 to perform mutation detection and generate a VCF file.

7. The method according to any one of claims 1 to 6, characterized in that: The reference genome in step (b) is determined according to the poultry species being tested.

8. The method according to any one of claims 1 to 7, characterized in that: The poultry is poultry with male chromosomes being ZZ and female chromosomes being ZW, including chickens, ducks and geese.

9. Use of the method according to any one of claims 1 to 8 in poultry sex identification.

10. Use of the variant sites captured by the method according to any one of claims 1 to 8 in bioinformatics analysis.

Images