A kit for screening lactation traits of Xinjiang donkeys and its application

Abstract

The invention discloses a kit for screening the lactation traits of Xinjiang donkeys and an application thereof. The kit for screening the lactation traits of Xinjiang donkeys is prepared by selecting a specific SNP site of Xinjiang donkey CA8 gene as a molecular marker, matching specific primers and a corresponding amplification system. Individuals with relatively high milk production and non-fat components and protein content in donkey milk can be accurately screened through detection, and individuals with excellent milk production shape of Xinjiang donkeys are selected and bred according to corresponding genotypes, thereby accelerating the breeding process and promoting the further development of the donkey industry.

Description

Technical Field

[0001] The invention relates to an identification method for animal genetic breeding, and specifically, the invention relates to a kit for screening the lactation traits of Xinjiang donkeys and the technical field of its application. Background Art

[0002] Donkey milk is actually an ancient product. Herodotus drank it as a nutritious beverage in the fifth century BC. In Egypt 2500 BC, there were also records about donkey milk on reliefs. In my country, Sun Simiao, the king of medicine in the Tang Dynasty, recorded in "Thousand Gold Food Treatment" that donkey milk "treats severe fever, jaundice, and quenches thirst". Li Shizhen, a medical scientist in the Ming Dynasty, also recorded in "Compendium of Materia Medica" that "donkey milk tastes sweet, cold, non-toxic, and is mainly used to treat children with acute fever and jaundice. Taking it frequently can relieve constipation, treat severe fever, stop thirst, children with acute fever, shock evil red heat, children's diseases, and cook it frequently with heat. It can treat qi evil, relieve children's heat toxins, and prevent smallpox." It is not difficult to see that donkey milk not only has a certain history, but also has certain medicinal value.

[0003] However, there is no specialized breed of donkeys for dairy production, and the individual milk production of female donkeys for production is low, far lower than cow milk production, and even lower than horse milk production, which cannot meet people's needs for donkey milk products. Xinjiang donkeys are a breed of livestock with multiple economic uses such as meat, leather, medicine, milk, and labor. Breeding dairy donkeys with high milk production performance can not only fill the market gap for donkey milk, but also significantly improve the feeding value and economic value of Xinjiang donkeys, which is an important task to promote the development of the donkey industry. Summary of the invention

[0004] In view of the technical status that there are no reports on accurate and effective molecular markers for screening lactation traits of Xinjiang donkeys in the prior art and there is no research on SNP markers specifically for CA8 gene in screening lactation traits of Xinjiang donkeys, the present invention aims to provide a detection kit containing CA8 gene for improving lactation traits of Xinjiang donkeys, and use the blood samples of Xinjiang donkeys as test materials. The detection kit of the present invention is used for breeding to improve lactation traits of Xinjiang donkeys to increase milk production, and it is expected to select DNA markers closely related to milk production traits, thereby accelerating the breeding process.

[0005] The invention provides a kit for screening the lactation trait of Xinjiang donkeys. The kit contains a SNP site g.89567442 for detecting a CA8 gene.

[0006] The invention provides a kit for screening the lactation trait of Xinjiang donkeys, wherein the allele type factors of the SNP site g.89567442 of the CA8 gene in the kit include GG, GT and TT.

[0007] The present invention provides a kit for screening the lactation traits of Xinjiang donkeys, wherein the kit is obtained by constructing the following co-construction system:

[0008] (1) According to the SNP site g.89567442 of the Xinjiang donkey CA8 gene, specific primers were designed manually;

[0009] (2) PCR reaction reagents: 2×KASPMix, upstream and downstream primers, DNA template and water.

[0010] The specific primer sequences are as follows:

[0011] Upstream primer 1: 5′-GAAGGTCGGAGTCAACGGATTGAAAAGGAGCAATACAATCACACTA-3′;

[0012] Upstream primer 2: 5′-GAAGGTGACCAAGTTCATGCTAAAAGGAGCAATACAATCACACTC-3′;

[0013] Downstream primer 1: 5'-CAACTCTTTTAGCCGATTATTTTATT-3'.

[0014] The present invention also provides an application of a kit for screening the lactation traits of Xinjiang donkeys. The application of the kit is embodied by a detection method, which specifically includes the following steps:

[0015] (1) Using a blood collection tube filled with sodium citrate anticoagulant, 5 ml of jugular vein blood was collected from a Xinjiang donkey and stored at -20°C; genomic DNA was extracted and stored at -20°C for later use;

[0016] (2) The genomic DNA of the Xinjiang donkey extracted in step (1) was used for PCR amplification of the CA8 gene. According to the designed primers, the CA8 gene was specifically amplified using the kit provided in any one of claims 1 or 2; the PCR reaction system was 2×KASP Mix 5 μL, 10.015 μL of 100 μM upstream primer, 20.015 μL of 100 μM upstream primer, 0.04 μL of 100 μM downstream primer, 0.07 μL of each upstream and downstream primer, 2 μL of genomic DNA, and ddH2O was added to 10 μL; the reaction conditions were: 94°C initial step, 1 cycle for 10 min; 94°C for 10 cycles for 20 s; 61°C for 45 s; 94°C for 35 cycles for 20 s; 55°C for 20 s;

[0017] (3) Perform KASP typing based on the product amplified in step (2): SNP typing is performed based on the specific matching of the primer terminal bases, and the Douglas platform is used to perform biallelic judgment on the SNP, thereby performing genotyping.

[0018] Furthermore, the present application also provides the use of a kit for screening lactation traits of Xinjiang donkeys in the breeding of Xinjiang donkeys with high milk production.

[0019] Furthermore, the present application also provides a kit for screening the lactation traits of Xinjiang donkeys for use in the breeding of Xinjiang donkeys with relatively high non-fat components and protein content.

[0020] In the present invention, in the design of the specific primers of CA8 gene of Xinjiang donkey, the DNA sequence of CA8 gene of donkey (GenBank ID: 106841674) was retrieved through the international molecular biological information website NCBI (National Center for Biotechnology), and the specific primers were designed manually.

[0021] By adopting the technical solution provided above, the present invention achieves the following beneficial effects:

[0022] (1) The present invention provides a kit for breeding the lactation trait of Xinjiang donkeys, and uses the kit to detect the SNP site g.89567442 of the CA8 gene of Xinjiang donkeys. The SNP site g.89567442 of the CA8 gene is closely related to the milk production (kg / d) of Xinjiang donkeys. The milk production of GG type in the late lactation period is significantly higher than that of TT (P < 0.01). In actual production applications, by selecting individuals with GG genotype and GT genotype, eliminating individuals with TT genotype, breeding to improve the milk production of Xinjiang donkeys, and used for the cultivation of specialized dairy donkey varieties, it has practical application value for protecting the excellent variety resources of Xinjiang donkeys, increasing the economic income of farmers and herdsmen, and accelerating the development of dairy donkeys.

[0023] (2) In the present invention, as described above, in constructing a detection kit for breeding the lactation traits of Xinjiang donkeys, the CA8 gene in the prior art is used as a candidate gene affecting the lactation traits of livestock and a kit is constructed. In the Xinjiang donkey population, the CA8 gene SNP site is related to the milk production of the Xinjiang donkey, and there are three genotypes: GG, GT, and TT. G is the dominant allele. Mutation at this site increases the milk production of the Xinjiang donkey, which provides a theoretical basis and technical support for molecular assisted breeding for breeding Xinjiang donkey varieties with excellent lactation traits.

[0024] (3) The present invention provides a kit for breeding the lactation trait of Xinjiang donkeys, and uses the kit to detect the SNP site g.89567442 of the CA8 gene of Xinjiang donkeys. The SNP site g.89567442 of the CA8 gene is closely related to the milk quality of Xinjiang donkeys, and the TT type significantly improves the non-fat components and protein content. In actual production applications, the milk quality of Xinjiang donkeys can be improved by selecting individuals with TT genotypes, which can be used for the cultivation of specialized dairy donkey varieties, and has practical application value for protecting the excellent variety resources of Xinjiang donkeys, increasing the economic income of farmers and herdsmen, and accelerating the development of dairy donkeys. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is the SNP site typing result of CA8 gene. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0027] Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0028] Unless otherwise specified in the following examples, the materials and reagents used can be purchased from commercial sources.

[0029] Example 1: Kit for breeding lactation traits of Xinjiang donkeys

[0030] The invention provides a kit for screening the lactation trait of Xinjiang donkeys. The kit contains a SNP site g.89567442 for detecting a CA8 gene.

[0031] The invention provides a kit for screening the lactation trait of Xinjiang donkeys, wherein the allele type factors of the SNP site g.89567442 of the CA8 gene in the kit include GG, GT and TT.

[0032] The present invention provides a kit for screening the lactation traits of Xinjiang donkeys, wherein the kit is obtained by constructing the following co-construction system:

[0033] (1) According to the SNP site g.89567442 of the Xinjiang donkey CA8 gene, specific primers were designed manually;

[0034] (2) PCR reaction reagents: 2×KASPMix, upstream and downstream primers, DNA template and water.

[0035] The specific primer sequences are as follows:

[0036] Upstream primer 1: 5′-GAAGGTCGGAGTCAACGGATTGAAAAGGAGCAATACAATCACACTA-3′;

[0037] Upstream primer 2: 5′-GAAGGTGACCAAGTTCATGCTAAAAGGAGCAATACAATCACACTC-3′;

[0038] Downstream primer 1: 5'-CAACTCTTTTAGCCGATTATTTTATT-3'.

[0039] The present invention also provides an application of a kit for screening the lactation traits of Xinjiang donkeys. The application of the kit is embodied by a detection method, which specifically includes the following steps:

[0040] (1) Using a blood collection tube filled with sodium citrate anticoagulant, 5 ml of jugular vein blood was collected from a Xinjiang donkey and stored at -20°C; genomic DNA was extracted and stored at -20°C for later use;

[0041] (2) The genomic DNA of the Xinjiang donkey extracted in step (1) was used for PCR amplification of the CA8 gene. According to the designed primers, the CA8 gene was specifically amplified using the kit provided in any one of claims 1 or 2; the PCR reaction system was 2×KASP Mix 5 μL, 10.015 μL of 100 μM upstream primer, 20.015 μL of 100 μM upstream primer, 0.04 μL of 100 μM downstream primer, 0.07 μL of each upstream and downstream primer, 2 μL of genomic DNA, and ddH2O was added to 10 μL; the reaction conditions were: 94°C initial step, 1 cycle for 10 min; 94°C for 10 cycles for 20 s; 61°C for 45 s; 94°C for 35 cycles for 20 s; 55°C for 20 s;

[0042] (3) Perform KASP typing based on the product amplified in step (2): SNP typing is performed based on the specific matching of the primer terminal bases, and the Douglas platform is used to perform biallelic judgment on the SNP, thereby performing genotyping.

[0043] Example 2: Application of a kit for breeding lactation traits of Xinjiang donkeys

[0044] 1. Test method:

[0045] (1) Acquisition of test materials

[0046] This experiment was conducted from March 7, 2021 to December 4, 2021 at the donkey farm in Yuepu Lake County, Kashgar Prefecture, Xinjiang, to collect data. The experiment selected 89 Xinjiang donkeys with 3-5 parities, one month after foaling, and normal lactation as research subjects for data collection in this experiment. All healthy female donkeys in this experiment were under the same feeding level. The experimental donkey groups were distributed in different pens for centralized feeding, and they were free to eat and drink water. The staff cleaned the troughs and troughs at a fixed time every day, and regularly disinfected the donkey pens to keep the pens clean. The components of the diet are corn stalks, concentrates, soybean meal, bran, corn, etc. The milkers milked in turn at 8 am and 8 pm every day (the milking time should be advanced or postponed according to seasonal changes, no more than one hour before and after). After milking, the female donkeys and foals were mixed in the circle for two hours for the foals to feed freely. After feeding, the foals were raised in separate circles.

[0047] This experiment uses the Saykin formula to calculate the daily milk production of Xinjiang donkeys during lactation. The Saykin formula is expressed as: W = G × 24 / h. W represents the total milk production within 24 hours, G is the total milk production in half a day, and h is the machine milking time within 24 hours. Blood was collected from the jugular vein at the end of the trial period.

[0048] (2) Acquisition of molecular markers

[0049] Based on the donkey resequencing results, the single nucleotide polymorphism (SNP) of the gene was selected, see Table 1. The relevant specific primers were obtained using the involved. The primer sequences are shown in Table 2. The PCR amplification volume was 10μL, 2×KASP Mix 5μL, 100μM Primer 10.015μL, 100μM Primer 2 0.015μL, 100μM Primer Common 0.04μL, genomic DNA 2μL, ddH2O supplemented to 10μL. PCR conditions were: 94℃ start step (1 cycle 10min), 94℃ 10 cycles 20s, 61℃ 45s, 94℃ 35 cycles 20s, 55℃ 20s. Genomic DNA was isolated using the DNA kit from GeneOnBioTech. Competitive allele-specific PCR (KASP TM ,LGCGenomics,Teddington,Middlesex,UK) genotyping was performed on the selected three SNPs for double allele identification, and the genotype data of 89 Xinjiang donkeys were output for statistical analysis. The typing results are shown in Table 3.

[0050] The genotyping results of LGCGenomics were analyzed using Douglas software of LGCGenomics (www.lgcgroup.com). The χ2 test (χ2) was used to determine whether the population was in Hardy-Weinberg equilibrium (HWE), and one-way ANOVA was used to perform genetic association analysis on polymorphic SNPs, and SPSS27.0 software was used for statistical analysis. Data are expressed as mean ± standard error.

[0051] Table 1: SNP loci selected for Xinjiang donkey genotyping study

[0052] Gene GeneID Allele replacement Location chromosome number CA8 106841674 G / T 89567442 12

[0053] Table 2: Primer information

[0054]

[0055] Table 3: List of polymorphic SNPs and genotype frequencies of Xinjiang donkey

[0056]

[0057] 2. Results:

[0058] 2.1 SNP results associated with milk production.

[0059] SNP significantly affects the milk production of Xinjiang donkeys, as shown in Table 4. CA8g.89567442T>G is associated with milk production, among which the wild genotype GG of CA8g.89567442T>G has higher milk production than the mutant TT in the late lactation period, suggesting that the difference in the late lactation period may be caused by CA8.

[0060] Table 4: SNPs associated with milk production in Xinjiang donkey breeds

[0061]

[0062] Note: In the same column, different letters indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05)

[0063] 2.2 SNP results related to milk components.

[0064] SNP significantly affects the composition of Xinjiang donkey milk (see Table 5). CA8g.89567442T>GTT significantly increased the non-fat components and protein content (P<0.05). This suggests that the difference in milk fat rate, total solid content and urea nitrogen in Xinjiang donkey milk may be caused by CA8.

[0065] Table 5: SNPs associated with milk production in Xinjiang donkeys (%)

[0066]

[0067]

[0068] As described above, the present invention can be well implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the present invention.

Claims

1. A kit for screening lactation traits of Xinjiang donkeys, characterized in that: The kit is used to detect the SNP site of the CA8 gene, and the allele type of the SNP site of the CA8 gene includes GG, GT and TT; the kit is obtained by the following co-construction system: (1) According to the SNP site of Xinjiang donkey CA8 gene, specific primers were designed manually; (2) PCR reaction reagents: 2×KASP Mix, upstream and downstream primers, DNA template and water; The specific primer sequences are as follows: Upstream primer 1: 5′-GAAGGTCGGAGTCAACGGATTGAAAAGGAGCAATACAATCACACTA-3′; Upstream primer 2: 5′-GAAGGTGACCAAGTTCATGCTAAAAGGAGCAATACAATCACACTC-3′; Downstream primer 1: 5′-CAACTCTTTTAGCCGATTATTTTATT-3′.

2. The use of a kit for screening the lactation traits of Xinjiang donkeys as claimed in claim 1, characterized in that: The application of the kit is embodied by a detection method, which specifically includes the following steps: (1) Using a blood collection tube filled with sodium citrate anticoagulant, 5 ml of jugular vein blood was collected from a Xinjiang donkey and stored at -20°C; genomic DNA was extracted and stored at -20°C for later use; (2) The genomic DNA of Xinjiang donkey extracted in step (1) was used to perform PCR amplification of CA8 gene. According to the designed primers, the kit provided in claim 1 was used to specifically amplify CA8 gene; the PCR reaction system was 5 μL of 2 × KASP Mix, 0.015 μL of 100 μM upstream primer 1, 0.015 μL of 100 μM upstream primer 2, 0.04 μL of 100 μM downstream primer, 0.07 μL of each upstream and downstream primer, 2 μL of genomic DNA, and ddH2O was added to 10 μL; the reaction conditions were: 94°C initial step, 1 cycle for 10 min; 94°C for 10 cycles for 20 s; 61°C for 45 s; 94°C for 35 cycles for 20 s; 55°C for 20 s; (3) Perform KASP typing based on the product amplified in step (2): SNP typing is performed based on the specific matching of the primer terminal bases, and the Douglas platform is used to perform biallelic judgment on the SNP, thereby performing genotyping.

3. Application of the kit for screening the lactation traits of Xinjiang donkeys as claimed in claim 1 in the breeding of Xinjiang donkeys with high milk production in the late lactation period.

4. Application of the kit for screening the lactation traits of Xinjiang donkeys as claimed in claim 1 in the breeding of Xinjiang donkeys with relatively high non-fat components and protein content.

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