Application of AAGAB gene molecular marker g.23443T>C in molecular marker-assisted breeding of Hu sheep

Abstract

The present invention belongs to the technical field of molecular marker-assisted breeding of Hu sheep, and specifically relates to the application of the AAGAB gene molecular marker g.23443T>C in molecular marker-assisted breeding of Hu sheep. The present invention uses Hu sheep as the research object, employs PCR amplification, direct product sequencing, and sequence analysis methods to analyze AAGAB gene polymorphisms, and comprehensively analyzes the correlation between different genotypes at the polymorphic sites and different growth traits. The analysis results show that g.23443T>C is significantly correlated with the chest circumference and daily weight gain of Hu sheep from six months of age to one year old. The birth weight, weaning weight, and six-month-old weight of individuals with the CC genotype are significantly higher than those with the TT and TC genotypes.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular marker-assisted breeding of Hu sheep, and in particular relates to the application of AAGAB gene molecular marker g.23443T>C in molecular marker-assisted breeding of Hu sheep. Background Art

[0002] The Huyang sheep is a rare breed of sheep in my country, native to the Taihu Lake. Its lambskin is renowned for its beautifully patterned fur. It is a unique breed of sheep used for lambskin production in my country and one of the few white lambskin breeds in the world. It enjoys a high reputation in the international market, earning it the nickname "soft gem." Huyang sheep boast delicious meat. Huyang sheep are large, high-yielding, and offer a rich, nutritious texture. Compared to other meats, Huyang meat is high in protein, low in fat, and low in cholesterol, resulting in a juicy texture and minimal mutton flavor. However, the meat quality of Huyang sheep differs significantly from that of other meat-producing breeds, such as the Dorper and German Merino. Therefore, selective breeding for growth performance is crucial to improve Huyang sheep's meat production.

[0003] The applicant has been authorized to use Chinese invention patents (publication numbers: CN109251986A, CN109182553A, CN109251987A, CN109251985A, CN109182554A and CN109055579A) to disclose candidate functional genes and SNPs for body size traits of Hu sheep screened using GWAS technology, and through population verification of SNPs, obtained SNPs that can be used for molecular marker-assisted breeding, which can be used for molecular marker-assisted breeding of Hu sheep meat traits and early selection of individuals in the core group of Hu sheep meat lines.

[0004] The applicant has also applied for Chinese invention patent applications (CN114381531A, CN114438232A, CN114574596A, and CN114480676A), which disclose 15 SNPs within the intron 1 region of the CAPN6 gene, four of which are significantly associated with the weight and body size traits of Hu sheep: 1. g.43756G>A is significantly associated with Hu sheep body length, with AA-type individuals showing significantly greater body length than GA-type individuals (P<0.05), while GG-type individuals show no significant difference from GA or AA-type individuals (P>0.05). 2. g.43815G>A is significantly associated with Hu sheep height, with AA-type individuals showing significantly greater height than GG-type individuals (P<0.05), while GA-type individuals show no significant difference from GG or AA-type individuals (P>0.05). 3. g.43851G>A was significantly correlated with body length of Hu sheep. The body length of AA-type individuals was significantly greater than that of GA-type individuals (P<0.05), while the difference between GG-type individuals and GA and AA-type individuals was not significant (P>0.05). 4. g.43917A>G was significantly correlated with body length and daily weight gain of Hu sheep. The body length of AA and GG-type individuals was significantly greater than that of AG-type individuals (P<0.05), while the difference between AA and GG was not significant (P>0.05). The daily weight gain of GG-type individuals from weaning to six months of age was significantly greater than that of AA-type individuals, while the difference between AG-type individuals and AA and GG was not significant.

[0005] In addition, the applicant has also applied for Chinese invention patent applications (CN117757958A, CN117701736A, CN117844943A, and CN117683908A) disclosing four SNPs within the intron 2 region of the ARHGAP24 gene, four of which are significantly associated with daily weight gain and / or annual weight of Hu sheep from six months to one year of age: 1. g.14979C>T is significantly associated with daily weight gain and annual weight of Hu sheep from six months to one year of age, with the daily weight and annual weight of individuals with the TT genotype being significantly higher than those with the CT and CC genotypes. 2. g.15042C>T is significantly associated with daily weight gain and annual weight of Hu sheep from six months to one year of age, with the daily weight and annual weight of individuals with the TT genotype being significantly higher than those with the CT and CC genotypes. 3. g.15255T>C was significantly correlated with daily weight gain from six months to one year of age and annual weight of Hu sheep. Individuals with the CC genotype had significantly higher daily weight gain from six months to one year of age and annual weight than those with the TC and TT genotypes. 4. g.15599G>T was significantly correlated with annual weight of Hu sheep. Individuals with the TT genotype had significantly higher daily weight gain from six months to one year of age and annual weight than those with the GT and GG genotypes.

[0006] AAGAB (Alpha And Gamma Adaptin Binding Protein) is a protein-coding gene consisting of 10 exons and corresponding introns, encoding the 315-amino acid residue α / γ adaptor binding protein p34 (α- and γ-adaptin binding protein p34). The AAGAB gene encodes two functional domains of the p34 protein: the N-terminal Rab-like GTPase domain encoding region is located in exons 2–4, and the C-terminal adaptor protein binding domain encoding region is located in exons 5–10. Mutations in AAGAB prevent the AP2 subunit from forming the adaptor complex, leading to AP2 subunit degradation and defective clathrin-mediated endocytosis, ultimately causing disease. Research on this gene has primarily focused on palmoplantar keratoderma (PPK), a condition characterized by abnormal thickening and hyperkeratosis of the skin on the palms or feet. Summary of the Invention

[0007] To study the correlation between AAGAB gene polymorphism and growth traits of Hu sheep and obtain genetic markers associated with growth traits, the present invention uses Hu sheep as research subjects, adopts PCR amplification, direct product sequencing, and sequence analysis methods to analyze AAGAB gene polymorphism, and comprehensively analyzes the correlation between different genotypes of polymorphic sites and different growth traits.

[0008] One object of the present invention is to provide a reagent for detecting a molecular marker that is significantly correlated with the chest circumference and daily weight gain of Hu sheep from six months of age to one year old. The molecular marker is located at g.23443T>C, located at the 264th position of SEQ ID NO:1, which is T or C; wherein: the chest circumference of individuals with CC genotype is significantly higher than that of individuals with TT genotype, and the daily weight gain of individuals with CC and TC genotypes from six months of age to one year old is significantly higher than that of individuals with TT genotype.

[0009] Furthermore, an object of the present invention is to provide a primer pair for identifying the molecular marker.

[0010] Preferably, the nucleotide sequences of the primer pair are shown as SEQ ID NO: 2 and SEQ ID NO: 3.

[0011] Furthermore, an object of the present invention is to provide a kit comprising the reagent or the primer pair.

[0012] Furthermore, an object of the present invention is to provide the use of the reagent, the primer pair or the kit in screening Hu sheep chest circumference and daily weight gain from six months to one year old to assist in breeding.

[0013] Furthermore, an object of the present invention is to provide an application of an amplified product in screening Hu sheep chest circumference and daily weight gain from six months to one year old for auxiliary breeding. The amplified product is obtained by amplification using the reagent, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0014] Furthermore, an object of the present invention is to provide a method for screening chest circumference and daily weight gain traits from six months of age to one year old, the method comprising the following steps: extracting genomic DNA of Hu sheep, performing PCR amplification using primer pairs, and detecting molecular markers in the amplified products, thereby screening the chest circumference and daily weight gain traits from six months of age to one year old of Hu sheep; the molecular marker is located as g.23443T>C, located at the 264th position of SEQ ID NO:1, which is T or C; wherein: the chest circumference of individuals with CC genotype is significantly higher than that of individuals with TT genotype, and the daily weight gain from six months of age to one year old of CC and TC genotypes is significantly higher than that of individuals with TT genotype.

[0015] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0016] Preferably, the PCR reaction system is 20.0-30.0 μL, KOD One™ PCR Master Mix -Blue 10.0-15.0 μL, 0.15-0.25 μL each of upstream and downstream primers, and 10.0-12.0 μL of sterile water.

[0017] Preferably, the PCR reaction program is: denaturation at 95-98°C for 8-15s, annealing at 60-65°C for 25-35s, extension at 65-70°C for 25-35s, reaction for 30-40 cycles, and storage at 0-5°C after the PCR is completed.

[0018] This study used Hu sheep as the research subjects, using PCR amplification, direct product sequencing, and sequence analysis to analyze AAGAB gene polymorphisms and comprehensively analyze the correlations between different genotypes at the polymorphic loci and various growth traits. The results showed that g.23443T>C was significantly correlated with chest circumference and daily weight gain from six months to one year of age. The birth weight, weaning weight, and six-month body weight of individuals with the CC genotype were significantly higher than those with the TT and TC genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 PCR amplification of SNPs detection primers for AAGAB gene of Hu sheep, M: DL2000.

[0020] Figure 2 Figure 2 is the nucleotide sequence of the amplified product of the present invention. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0022] 1 Materials and Methods

[0023] 1.1 Sample collection

[0024] Blood samples were collected from the jugular vein of 133 Hu sheep (ewes), anticoagulated with EDTA, and stored at -20°C. Samples were collected from Hangzhou Pangda Agricultural Development Co., Ltd. Growth traits measured included height, length, chest circumference, weaning weight, six-month weight, and one-year-old weight.

[0025] 1.2 Primer design

[0026] PCR amplification primers were designed using DNAMAN 8.0 software based on the sheep AAGAB gene sequence (Acc: 25662) published by Ensembl. The primer sequences are shown in Table 1. The primers were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0027] Table 1 PCR amplification primers for Hu sheep AAGAB gene

[0028]

[0029] 1.3 PCR amplification and sequence analysis

[0030] A 25 μL PCR reaction system was used: 12.5 μL KOD One™ PCR Master Mix -Blue, 0.2 μL each of the upstream and downstream primers (10 μM), and 11.1 μL sterile water. PCR reaction conditions were: denaturation at 98°C for 10 s, annealing at 61°C for 30 s, and extension at 68°C for 30 s, for 34 cycles. After completion of PCR, the reaction mixture was stored at 4°C.

[0031] The amplified products were sent to Anhui General Biotechnology for Sanger sequencing. The sequencing peaks of each individual were analyzed using Mutation Surveyor 5.02 (Softgenetics, USA) software to determine the location and mode of mutation in each individual.

[0032] 1.4 Data Statistics and Analysis

[0033] 1.4.1 Polymorphic Information Content Analysis (PIC)

[0034] The PIC value was calculated using little Programe, and the site heterozygosity, Shannon information content, gene frequency, and genotype frequency of each SNP were calculated using PopGen 32 software.

[0035] Polymorphism information content (PIC) analysis is used to determine and analyze the information content expressed by a genetic marker. It indicates the likelihood that an offspring inherits a particular allele from the same allele in its parent and is an ideal indicator for measuring allelic segment polymorphism. A PIC greater than 0.5 indicates a highly polymorphic locus, 0.25 less than PIC less than 0.5 indicates a moderately polymorphic locus, and PIC less than 0.25 indicates a lowly polymorphic locus. A higher PIC value indicates a greater number of effective alleles and a greater degree of heterozygosity.

[0036] The calculation formula is:

[0037]

[0038] P i and P j are the frequencies of the ith and jth alleles, respectively; n is the number of alleles.

[0039] Site heterozygosity (He) refers to the average frequency of heterozygous individuals at each locus. Heterozygosity objectively reflects the level of genetic variation within a population. A higher average heterozygosity value indicates greater genetic variation within the population, richer genetic diversity, greater genetic potential, and better application in animal genetic breeding research. A lower value indicates greater genetic consistency, indicating less genetic variation within the population and lower genetic potential. The calculation formula is:

[0040]

[0041] p i represents the frequency of the ith allele.

[0042] Shannon Information Content

[0043] SIC (shannon information content), calculated as:

[0044] SIC=-C*logP i

[0045] Where: P i is the frequency of the ith allele in the population, and C is a constant.

[0046] Gene frequency and genotype frequency

[0047] ①Gene frequency = number of individuals with the same genotype / number of individuals in the population × 100%

[0048] ②Gene frequency = homozygous genotype frequency + 1 / 2 × heterozygous genotype frequency

[0049] 1.4.2 Correlation Analysis

[0050] The general linear model (GLM; SPSS) was used to explore SNPs associated with the weight and body size traits of Hu sheep.

[0051] Since all individuals analyzed were from the same farm, under the same rearing environment and management conditions, and were all ewes, farm effects and sex effects were not included in the data modeling.

[0052] The specific model is: Y=Xβ+e

[0053] Where Y: phenotypic value vector of body size and weight traits of Hu sheep;

[0054] β: fixed effect vector of phenotypic mean, SNP, etc.;

[0055] e: residual effect vector;

[0056] X is the incidence matrix of β.

[0057] 2 Test results

[0058] 2.1 PCR amplification results of SNPs detection in Hu sheep AAGAB gene

[0059] The PCR product is brighter, with a single band and no non-specific amplification ( Figure 1 ), the actual PCR product is consistent with the expected PCR amplification product size, and subsequent PCR product direct sequencing can be performed.

[0060] 2.2 Sequencing results analysis

[0061] The sequencing results were compared using Mutation Surveyor 5.02 software, and two SNPs were found in the AAGAB gene, namely g.23366T>C and g.23443T>C, both of which had three genotypes, namely TT / TC / CC and TT / TC / CC.

[0062] Table 2 The position of mutation sites in the amplified sequence

[0063]

[0064] 2.3 Population genetics analysis of the AAGAB gene

[0065] Table 3 shows that the effective alleles are 1.07 and 1.48, the average heterozygosity is 0.07 and 0.32, and the Shannon information content is 0.15 and 0.50, respectively. g.23366T>C belongs to low polymorphism (PIC<0.25), and g.23443T>C belongs to moderate polymorphism (0.25<PIC<0.50).

[0066] Table 3 Population genetics analysis of AAGAB gene SNP loci

[0067]

[0068] 2.4 Association analysis between AAGBA gene SNPs and growth traits

[0069] Using the association analysis of SNPs of AAGAB gene and growth traits of Hu sheep in “1.4.2 Association Analysis”, as shown in Table 4, g.23443T>C is significantly correlated with chest circumference and weight from six months to one year old of Hu sheep ( P <0.05), the chest circumference of CC genotype individuals was significantly higher than that of TT genotype individuals, and there was no significant difference between CC genotype and TC genotype. The daily weight gain of CC and TC genotypes from six months to one year old was significantly higher than that of TT genotype individuals.

[0070] Table 4 Association analysis between AAGAB gene SNPs and Hu sheep growth traits

[0071]

[0072] Note: Data with different superscript letters in the same column and position are significantly different (P < 0.05).

[0073] 3. Result analysis:

[0074] 3.1 Correlation analysis between SNP locus g.23366T>C and growth traits of Hu sheep

[0075] The statistical significance level (P value) for g.23366T>C was greater than 0.05 for each trait, indicating that this locus has no significant effect on Hu sheep growth traits in this sample population. Differences between genotypes (TT, TC, CC) in body height, chest circumference, body length, weaning weight, and weight growth rate were small and not statistically significant. Although individuals with the CC genotype predominated in the sample (n=126), their average weight growth was slightly higher than that of the TT / TC genotype at several stages, the differences were not significant. Therefore, g.23366T>C is not a suitable target locus for marker-assisted breeding.

[0076] 3.2 Correlation analysis between SNP site g.23443T>C and growth traits of Hu sheep

[0077] 1) Chest Circumference (cm): P = 0.04, reaching a significant difference. The CC genotype (100.20±6.58) had a significantly higher value than the TT genotype (94.50±5.78), while the TC genotype (99.07±6.99) was intermediate. The letters (a, b) also indicate CC > TC > TT, indicating statistically significant stratification.

[0078] 2.) Daily weight gain from six months to one year old (kg):

[0079] P = 0.04, also reaching the significance level. Both the CC and TC genotypes had significantly higher rates than the TT genotype (0.17a vs 0.12b kg / day), but there was no significant difference between CC and TC, suggesting the possibility of a dominant genetic effect.

[0080] 3) Other traits (height, length, birth weight, weaning weight, daily weight gain from weaning to six months of age, yearling weight, etc.)

[0081] The P values ​​were all greater than 0.05, indicating that there was no significant statistical difference. However, it is worth noting that the CC type was at a better level in multiple indicators (such as birth weight and body length), which has potential reference value.

[0082] 3.3 Summary of the application value of molecular breeding

[0083] The g.23443T>C SNP is the only SNP found in this study to be significantly associated with growth traits (chest circumference and growth rate). Individuals with the CC / TC genotype exhibit superior growth potential. This locus exhibits moderate polymorphism (PIC = 0.50) and possesses population screening efficacy, making it a promising molecular discriminant marker for core breeding populations. This SNP can be used to develop commercial molecular breeding tools, such as SNP detection kits, to guide farms in early identification of high-quality breeding sheep.

[0084] The above is a description of the embodiments of the present invention. The above description of the disclosed embodiments will enable professionals in the field to implement or use the present invention. Various modifications to these embodiments will be apparent to professionals in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A reagent for detecting molecular markers for use in assisted breeding for screening Hu sheep chest circumference and daily weight gain from six months to one year old; characterized in that: The molecular marker is located at the 264th position of SEQ ID NO: 1, which is T or C; among them: the chest circumference of the CC genotype is significantly higher than that of the TT genotype individual, and the daily weight gain of the CC and TC genotypes from six months of age to one year old is significantly higher than that of the TT genotype individual.

2. Application of primer pairs in assisted breeding for screening Hu sheep chest circumference and daily weight gain from six months to one year old; characterized in that, The molecular marker used for identification by the primer pair is located at position 264 of SEQ ID NO: 1, which is T or C; among them: the chest circumference of individuals with CC genotype is significantly higher than that of individuals with TT genotype, and the daily weight gain of CC and TC genotypes from six months of age to one year old is significantly higher than that of individuals with TT genotype.

3. The use according to claim 2, characterized in that The nucleotide sequences of the primer pair are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

4. A kit comprising a primer pair for use in assisted breeding for screening Hu sheep chest circumference and daily weight gain from six months to one year old; characterized in that: The primer pair is a primer pair for identifying a molecular marker; the molecular marker is located at position 264 of SEQ ID NO: 1, which is T or C; wherein: the chest circumference of individuals with the CC genotype is significantly higher than that of individuals with the TT genotype, and the daily weight gain from six months of age to one year of age of the CC and TC genotypes is significantly higher than that of individuals with the TT genotype.

5. A method for screening individuals of Hu sheep for chest circumference and daily weight gain from six months to one year old, characterized in that: The method comprises the following steps: extracting Hu sheep genomic DNA, performing PCR amplification using a molecular marker detection primer pair, and detecting molecular markers in the amplified product, thereby screening the Hu sheep for chest circumference and daily weight gain traits from six months of age to one year of age; the molecular marker is located at the 264th position of SEQ ID NO: 1, which is T or C; wherein: the chest circumference of individuals with the CC genotype is significantly higher than that of individuals with the TT genotype, and the daily weight gain of individuals with the CC and TC genotypes from six months of age to one year of age is significantly higher than that of individuals with the TT genotype.

6. The method according to claim 5, characterized in that The nucleotide sequences of the primer pair are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

7. The method according to claim 5, characterized in that The PCR reaction system is 20.0-30.0 μL, KOD One™ PCR Master Mix -Blue 10.0-15.0 μL, 0.15-0.25 μL of upstream and downstream primers, and 10.0-12.0 μL of sterile water.

8. The method according to claim 6 or 7, characterized in that The PCR reaction program was as follows: denaturation at 95-98°C for 8-15s, annealing at 60-65°C for 25-35s, and extension at 65-70°C for 25-35s, for 30-40 cycles, and storage at 0-5°C after completion of PCR.

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