A SNP molecular marker for identifying the gender of sheep and application thereof

Abstract

The application discloses a SNP molecular marker for identifying the gender of sheep and application thereof. The SNP molecular marker comprises one of SNP1, SNP2, SNP3, SNP4 and SNP5. The application provides a SNP molecular marker for identifying the gender of sheep, and the SNP molecular marker has high typing quality, single copy and high sample detection rate, and can be used for molecular marker assisted breeding of the gender of sheep. The SNP molecular marker can realize early gender typing identification of sheep embryos in a quick, high-throughput and low-cost manner, and the gender identification can be completed before embryo transplantation, so that pregnancy, delivery and feeding consumption of non-target embryos can be effectively avoided, and the breeding cycle can be directly shortened, and the selection efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an SNP molecular marker for identifying the sex of sheep and its application. Background Technology

[0002] Through long-term domestication, natural selection, and artificial selection, sheep germplasm resources have developed into a highly diverse group. Sheep breeds can be broadly categorized by their primary use into meat, wool, fur, and dairy types. Notable breeds include the Australian Merino, renowned for its fine wool; the Dorper and Suffolk, excelling in meat production; Mongolian sheep; Tibetan sheep; Kazakh sheep; Small-tailed Han sheep; Hu sheep; Tan sheep; Ujumqin sheep; and Xinjiang fine-wool sheep. The development of the sheep industry primarily relies on the precise identification and utilization of germplasm resources, as well as the intensive farming methods and the extension of the entire industry chain.

[0003] Sex selection in sheep farming is a crucial factor influencing breeding efficiency, reproductive management, and production direction. In breeding-oriented sheep farming, ewes are the foundation of reproduction and directly impact flock growth rate; therefore, ewes should be prioritized, making up over 90% of the flock, with only a small number of high-quality rams needed. In meat-oriented sheep farming, male lambs are preferred, as they typically grow faster than ewes, have higher feed conversion rates, and are better suited for short-term fattening. In dairy-oriented sheep farming, ewes are prioritized, as they are the primary milk producers, and rams are usually sold for meat. Therefore, scientifically planned sex selection in sheep farming can significantly improve the economic efficiency and sustainability of sheep farming.

[0004] The sex inheritance mechanism in sheep is consistent with that of mammals, following an XY sex determination system. Sheep possess both X and Y chromosomes, and sex is determined by the sex chromosome (X or Y) carried by the paternal sperm. The SRY gene, located in the sex-determining region of the Y chromosome, has been identified as a key gene triggering testicular differentiation. If the embryo expresses the SRY protein, the gonads develop into testes; otherwise, they differentiate into ovaries. In sheep reproduction, sex control is primarily achieved through two methods: X / Y sperm separation and early embryonic sex determination. Sex control can be achieved through sperm separation technology. Flow cytometry can separate X / Y sperm, which, combined with artificial insemination or in vitro fertilization, can significantly increase the probability of offspring of a specific sex. However, sperm cell separation technology is complex and costly, and sheep sperm separation techniques can affect sperm motility, acrosome integrity, and mitochondrial activity to varying degrees, making it difficult to apply in large-scale production. Therefore, early embryonic sex determination has become the main method for sex control in sheep. Currently, animal embryo transfer technology has been widely used in sheep farming. If the sex of early sheep embryos can be accurately and easily identified, and embryos of known sex can be transferred as needed, the supply and demand contradiction of sheep can be resolved, the economic benefits of sheep farming can be improved, and there is a broad market prospect.

[0005] Early embryonic sex determination is an important aspect of livestock embryo transfer technology and one of the main methods for controlling the sex of livestock offspring. With the development of embryo transfer technology, early embryonic sex determination has become one of the most practical sex control technologies. Various methods exist for embryonic sex determination. Early methods for mammalian sex determination used PCR reaction systems, involving the amplification of the SRY gene (a unique sequence located on the Y chromosome). However, the SRY gene only displays one band; if the SRY gene band does not appear during the experiment, it is impossible to determine whether the sample is female or if an error occurred during the experiment. Although genes present in both females and males (such as ZFX / Y) can be used as positive controls, this requires increased time and materials. There are also reports of using the AMEL gene for sex determination. The AMEL gene exists in homologous regions on both the X and Y chromosomes; based on the different intron lengths on the two sex chromosomes, gene fragments of different lengths can be amplified, which can be used for sex determination. These identification techniques are all based on PCR amplification and electrophoresis detection, with complex procedures that cannot achieve high-throughput detection.

[0006] At present, sheep sex identification is mainly done by judging the morphology of external genitalia after birth. The main disadvantages of this method are: (1) the identification time is delayed and it needs to be completed after the lamb is born; (2) the lamb of the non-target sex has consumed all the resources of embryonic development, maternal pregnancy, parturition and early feeding; resulting in a double waste of feed and labor costs; leading to a longer breeding cycle of superior breeds and making it impossible to expand the core population in a targeted manner. The second method is fetal identification during pregnancy, including B-ultrasound and amniocentesis. The main disadvantages of this method are: (1) it requires more than 45 to 60 days of pregnancy to make an accurate judgment, the identification time is still too late and depends on the technical level of the operator; (2) if the identified sex is not the target sex, the pregnancy needs to be terminated, which causes physiological damage to the mother, reduces the subsequent reproductive capacity of the recipient ewe, and there is a certain surgical risk in terminating the pregnancy, which may cause maternal infection; (3) amniocentesis is an invasive operation that is easy to damage the embryo, which increases the risk of embryonic abortion by 3% to 5%; and the detection cost is high, making it difficult to apply on a large scale; especially rare breeds have a high risk of embryo loss, which is not conducive to the protection of germplasm resources. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an SNP molecular marker for identifying the sex of sheep.

[0008] The present invention also proposes a primer set for detecting the above-mentioned SNP molecular markers.

[0009] The present invention also proposes a reagent kit.

[0010] This invention also proposes the application of the above-mentioned SNP molecular markers, primer sets, and / or kits.

[0011] This invention also proposes a method for identifying or assisting in the identification of sex.

[0012] This invention also proposes a sheep breeding method.

[0013] According to a first aspect of the present invention, a SNP molecular marker for identifying the sex of sheep is provided, said SNP molecular marker including one of SNP1, SNP2, SNP3, SNP4 and SNP5; The SNP1 is located at position 52665376 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, and the polymorphism is C / T; The SNP2 is located at position 83519215 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of C / A. The SNP3 is located at position 97628322 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of A / G; The SNP4 is located at position 101569711 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of G / A; The SNP5 is located at position 141538303 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of C / T.

[0014] In some embodiments of the present invention, the sheep includes at least one of Mongolian sheep, Tibetan sheep, Kazakh sheep, Small-tailed Han sheep, Hu sheep, Tan sheep, Ujumqin sheep, Xinjiang fine-wool sheep, Australian Merino sheep, Dorper sheep, and Suffolk sheep.

[0015] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular markers is provided.

[0016] In some embodiments of the present invention, the primer set includes: (1) A primer set for amplifying SNP1, comprising specific primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; or, (2) A primer set for amplifying SNP2, comprising specific primers with sequences as shown in SEQ ID NO.4 and SEQ ID NO.5; or, (3) A primer set for amplifying SNP3, comprising specific primers with sequences as shown in SEQ ID NO.7 and SEQ ID NO.8; or, (4) A primer set for amplifying SNP4, comprising specific primers with sequences as shown in SEQ ID NO.10 and SEQ ID NO.11; or, (5) Primer set for amplifying SNP5, containing specific primers with sequences as shown in SEQ ID NO.13 and SEQ ID NO.14.

[0017] In some embodiments of the present invention, the primer set further includes universal primers with sequences such as SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 or SEQ ID NO.15.

[0018] In some embodiments of the present invention, the specific primers are respectively connected to different fluorescent adapters.

[0019] In some embodiments of the present invention, the fluorescent connector is selected from FAM and HEX.

[0020] According to a third aspect of the present invention, a kit is provided, the kit comprising the above-described primer set.

[0021] According to a fourth aspect of the present invention, the application of the above-described SNP molecular markers, primer sets, kits, or gene chips in any of the following is proposed: 1) To detect or assist in the detection of sheep sex; 2) Prepare products for detecting or assisting in the detection of sheep sex; 3) Select and breed ewes or rams; 4) Prepare products from selected ewes or rams; 5) Sheep breeding; 6) Prepare products for sheep breeding.

[0022] In some embodiments of the present invention, the sheep includes at least one of Mongolian sheep, Tibetan sheep, Kazakh sheep, Small-tailed Han sheep, Hu sheep, Tan sheep, Ujumqin sheep, Xinjiang fine-wool sheep, Australian Merino sheep, Dorper sheep, and Suffolk sheep.

[0023] According to a fifth aspect of the present invention, a method for identifying the sex of sheep using the above-mentioned molecular markers is provided, the method comprising the following steps: S1. Extract genomic DNA from the sheep sample to be tested; S2. Perform polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and determine the sex of the sheep to be tested based on the genotype.

[0024] In some embodiments of the present invention, the sheep sample to be tested includes sheep embryonic cells, blood, hair follicles, or any sheep tissue.

[0025] In some embodiments of the present invention, the criteria for determining the sex of the sheep to be tested based on genotype are as follows: When the SNP molecular marker is SNP1, if the genotype obtained by SNP1 detection is CT or CC, the sheep to be tested is a female sheep; if the genotype obtained by detection is TT, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP2, if the genotype obtained by SNP2 detection is AA or CA, the sheep to be tested is a female sheep; if the genotype obtained by detection is CC, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP3, if the genotype obtained by SNP3 detection is AA or AG, the sheep to be tested is a female sheep; if the genotype obtained by detection is GG, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP4, if the genotype obtained by SNP4 detection is GG or GA, the sheep to be tested is a female sheep; if the genotype obtained by detection is AA, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP5, if the genotype obtained by SNP5 detection is CC or CT, the sheep to be tested is a female sheep; if the genotype obtained by detection is TT, the sheep to be tested is a male sheep.

[0026] In some embodiments of the present invention, in step S2, the SNP molecular marker is detected using KASP (competitive allele-specific PCR) technology.

[0027] In some embodiments of the present invention, the composition of the KASP reaction mixture for detecting SNP molecular markers using KASP technology is as follows:

[0028] In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using KASP technology is as follows: 92-95℃ for 13-17 min; 92-95℃ for 18-22 s, 65℃-57℃ for 55-65 s, 8-12 cycles; 92-95℃ for 18-22 s, 55-58℃ for 55-65 s, 30-35 cycles.

[0029] In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using KASP technology is as follows: 94℃ for 15 min; 94℃ for 20 s, 65℃-57℃ for 60 s, 10 cycles; 94℃ for 20 s, 57℃ for 60 s, 33 cycles.

[0030] According to a sixth aspect of the present invention, a sheep breeding method is provided, comprising the following steps: selecting ewes or rams for subsequent breeding using the above-mentioned SNP molecular marker identification method.

[0031] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention provides a SNP molecular marker for identifying the sex of sheep, which can be efficiently used for sex identification in the early stages of sheep embryos, specifically including: (1) identification is completed before embryo transfer, which can effectively avoid pregnancy, parturition, and feeding consumption of non-target embryos, directly shorten the breeding cycle, and improve breeding efficiency; (2) the detection speed is fast, the automation throughput is high, the cost is low, and it is suitable for large-scale breeding; (3) through the genotype detection results, early selection can be achieved in the lambing stage, which greatly shortens the breeding cycle, promotes the transformation of the sheep industry from "experience-based breeding" to "molecular design breeding", and improves the overall economic benefits. At the same time, the SNP molecular marker has high typing quality, single copy, and high sample detection rate (>99%).

[0032] This invention provides a KASP-based SNP detection method for sheep sex determination. Through genotyping results, sex identification and selection can be performed in the early embryonic stage, significantly shortening the breeding cycle. This method is simple, highly automated, high-throughput, and fast, requiring minimal reagents and inexpensive. The results are accurate, reproducible, and stable, and can be used for sex determination in different sheep breeds, making it suitable for large-scale breeding testing. Furthermore, this method uses a small amount of reagents (only 0.8 μL / reaction), reducing reagent and consumable costs by 70%-90% compared to traditional 96-well plate SNP genotyping methods.

[0033] Furthermore, the SNP detection method of this invention is based on SNP labeling, possessing all the inherent advantages of SNP labeling. It boasts high labeling specificity, sensitivity, and resolution; it is unaffected by environmental conditions, can use embryonic cells, blood, hair follicles, or any tissue, and provides accurate, repeatable, and stable detection results with broad applicability. All the technology, reagents, consumables, and instruments used in this invention are domestically produced, unaffected by domestic or international environments, enabling more stable and reliable widespread adoption. Attached Figure Description

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a flowchart of the molecular marker screening and verification experiment in Example 1 of the present invention; Figure 2 This is a graph showing the results of a genome-wide association analysis in Example 1 of the present invention; Figure 3 This is a typing diagram of the OV900024 molecular marker in Example 1 of the present invention; Figure 4 This is a typing diagram of the OV900025 molecular marker in Example 1 of the present invention; Figure 5 This is a typing diagram of the OV900026 molecular marker in Example 1 of the present invention; Figure 6 This is a typing diagram of the OV900027 molecular marker in Example 1 of the present invention; Figure 7 This is a typing diagram of the OV900028 molecular marker in Example 1 of the present invention. Detailed Implementation

[0035] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Example 1: A SNP molecular marker for identifying the sex of sheep This embodiment provides a SNP molecular marker for identifying the sex of sheep. The specific screening and verification process is as follows: Figure 1 As shown, the method is as follows: 1. Mining of sex-related gene loci in sheep Blood samples were collected from 290 Kazakh sheep (including 44 rams and 246 ewes) and DNA was extracted for sheep 50K DNA testing. c GPS liquid-phase chip detection yielded genotypic data for 50,900 SNP loci. Genome-wide association analysis was performed combining genotypic and phenotypic data, with results as follows: Figure 2 As shown in the figure, most SNP sites on the X chromosome are sex-related in sheep.

[0037] 2. Label design and synthesis For the SNP loci obtained above, KASP marker primers were designed based on the sheep reference genome ARS-UI_Ramb_V2.0 using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). Each marker group had three primers, with two specific primers having FAM and HEX fluorescent sequences attached to their 5' ends, respectively. After design, genome-wide copy number analysis was performed on the primer sequences, ultimately resulting in five high-quality single-copy KASP marker loci. The locus information is shown in Tables 1-2, and the primers used for KASP marker detection are shown in Table 2.

[0038] When the genotype obtained from KASP marker OV900024 is CT or CC, the sheep being tested is a female; if the genotype is TT, the sheep is a male. When the genotype obtained from KASP marker OV900025 is AA or CA, the sheep being tested is a female; if the genotype is CC, the sheep is a male. When the genotype obtained from KASP marker OV900026 is AA or AG, the sheep being tested is a female; if the genotype is GG, the sheep is a male. When the genotype obtained from KASP marker OV900027 is GA or GG, the sheep being tested is a female; if the genotype is AA, the sheep is a male. When the genotype obtained from KASP marker OV900028 is CC or CT, the sheep being tested is a female; if the genotype is TT, the sheep is a male.

[0039] Table 1

[0040] Table 2

[0041] 3. Detection and verification of the markers Samples from 289 sheep of known sex (different from the 290 Kazakh sheep samples mentioned above) were collected for testing and verification. The verification and detection of KASP markers were performed using the Douglas Array Tape system. This genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0042] PCR amplification system: The PCR amplification system was automatically assembled using NEXAR, and the PCR amplification system is shown in Table 3 below.

[0043] Table 3 PCR amplification system for KASP marker genotyping

[0044] PCR amplification: PCR amplification was performed using SOELLEX under the following conditions: 94℃ for 15 minutes; 94℃ for 20 seconds, 65℃-57℃ (annealing temperature decreased by 0.8℃ per cycle) for 60 seconds, 10 cycles; 94℃ for 20 seconds, 57℃ for 60 seconds, 33 cycles.

[0045] Signal scanning and genotyping: After the PCR reaction, the fluorescence signal of the reaction products was scanned using ARAYA; then, genotyping and data analysis were performed using INTELLICS. In the above five KASP marker genotyping tests, the genotypes of the samples could be divided into three clusters: the X cluster, the Y cluster, and the heterozygous genotype cluster (see...). Figure 3-7 (As shown). The X cluster indicates that the sample contains a homozygous X allele at this KASP marker locus (marked in red in the upper left corner of the genotyping graph), the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker locus (marked in blue in the lower right corner of the genotyping graph), and the heterozygous genotype cluster indicates that the sample contains heterozygous X and Y alleles at this KASP marker locus (marked in purple in the genotyping graph).

[0046] Quality Validation of KASP Marker Genotyping for Sheep Sex Identification: Five KASP markers were used to test and validate 289 sheep samples of known sex. The proportions of rams and ewes for each marker's three genotypes were analyzed. The results are shown in Table 4. The table shows that all five KASP markers can be used to distinguish sheep sex, with marker OV900028 showing the best results. Homozygous C:C and heterozygous C:T genotypes of marker OV900028 were predominantly found in ewes, with proportions of 88% and 100%, respectively, while the homozygous T:T genotype was found in 95% of rams. This marker can effectively distinguish between rams and ewes. The results indicate that a small number of cells can be taken early in sheep embryo culture and tested using marker OV900028 for rapid screening of embryos of the desired sex for transplantation.

[0047] Table 4

[0048] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A SNP molecular marker for identifying the sex of sheep, characterized in that, The SNP molecular markers include one of SNP1, SNP2, SNP3, SNP4 and SNP5; The SNP1 is located at position 52665376 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, and the polymorphism is C / T; The SNP2 is located at position 83519215 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of C / A. The SNP3 is located at position 97628322 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of A / G; The SNP4 is located at position 101569711 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of G / A; The SNP5 is located at position 141538303 on the X chromosome of the sheep reference genome ARS-UI_Ramb_V2.0 version, with a polymorphism of C / T.

2. A primer set for amplifying the SNP molecular marker as described in claim 1.

3. The primer set according to claim 2, characterized in that, The primer set includes: (1) A primer set for amplifying SNP1, comprising specific primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; or, (2) A primer set for amplifying SNP2, comprising specific primers with sequences as shown in SEQ ID NO.4 and SEQ ID NO.5; or, (3) A primer set for amplifying SNP3, comprising specific primers with sequences as shown in SEQ ID NO.7 and SEQ ID NO.8; or, (4) A primer set for amplifying SNP4, comprising specific primers with sequences as shown in SEQ ID NO.10 and SEQ ID NO.11; or, (5) Primer set for amplifying SNP5, containing specific primers with sequences as shown in SEQ ID NO.13 and SEQ ID NO.

14.

4. The primer set according to claim 3, characterized in that, The primer set also includes universal primers with sequences such as SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 or SEQ ID NO.

15.

5. A reagent kit, characterized in that, The kit includes the primer set as described in any one of claims 2-4.

6. The use of the SNP molecular marker of claim 1, the primer set of any one of claims 2-4, the kit of claim 5, or the gene chip of claim 6 in any one of the following: 1) To detect or assist in the detection of sheep sex; 2) Prepare products for detecting or assisting in the detection of sheep sex; 3) Select and breed ewes or rams; 4) Prepare products from selected ewes or rams; 5) Sheep breeding; 6) Prepare products for sheep breeding.

7. The application according to claim 6, characterized in that, The sheep include at least one of the following: Mongolian sheep, Tibetan sheep, Kazakh sheep, Small-tailed Han sheep, Hu sheep, Tan sheep, Ujumqin sheep, Xinjiang fine-wool sheep, Australian Merino sheep, Dorper sheep, and Suffolk sheep.

8. A method for identifying the sex of sheep using the molecular markers described in claim 1, characterized in that, The method includes the following steps: S1. Extract genomic DNA from the sheep sample to be tested; S2. Perform polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and determine the sex of the sheep to be tested based on the genotype.

9. The method according to claim 8, characterized in that, The criteria for determining the sex of the sheep to be tested based on genotype are as follows: When the SNP molecular marker is SNP1, if the genotype obtained by SNP1 detection is CT or CC, the sheep to be tested is a female sheep; if the genotype obtained by detection is TT, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP2, if the genotype obtained by SNP2 detection is AA or CA, the sheep to be tested is a female sheep; if the genotype obtained by detection is CC, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP3, if the genotype obtained by SNP3 detection is AA or AG, the sheep to be tested is a female sheep; if the genotype obtained by detection is GG, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP4, if the genotype obtained by SNP4 detection is GG or GA, the sheep to be tested is a female sheep; if the genotype obtained by detection is AA, the sheep to be tested is a male sheep. When the SNP molecular marker is SNP5, if the genotype obtained by SNP5 detection is CC or CT, the sheep to be tested is a female sheep; if the genotype obtained by detection is TT, the sheep to be tested is a male sheep.

10. A sheep breeding method, characterized in that, The method includes the following steps: using the method of claim 8 or 9, selecting ewes or rams for subsequent breeding.

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