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

Abstract

The invention belongs to the technical field of Hu sheep molecular marker-assisted breeding, and particularly relates to application of an AAGAB gene molecular marker g.23443T > C in Hu sheep molecular marker-assisted breeding. According to the invention, Hu sheep is taken as a research object, PCR amplification, direct sequencing of products and sequence analysis are adopted, AAGAB gene polymorphism is analyzed, and correlation between different genotypes of polymorphic sites and different growth traits is comprehensively analyzed. An analysis result shows that g.23443T > C is remarkably related to the chest circumference of Hu sheep and the daily gain of Hu sheep from six-month-old to one-year-old, and the birth weight, weaning weight and six-month-old body weight of CC genotype are remarkably higher than those of TT and TC genotype individuals.

Description

Technical Field

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

[0002] Hu sheep is a rare sheep breed in China, originally from the Taihu Lake area. It is famous for its beautiful lambskin patterns. It is a unique lambskin sheep breed in China and also one of the few white lambskin breeds in the world at present. It enjoys a high reputation in the international market and is known as the "soft gem". Hu sheep has delicious meat, a large body size, high meat production, delicious meat, and rich nutrition. Compared with other meats, Hu sheep meat has the advantages of high protein, low fat, and low cholesterol, being juicy and having little mutton smell. However, there are still significant differences in the meat production performance of Hu sheep compared with foreign meat sheep breeds such as Dorper sheep and German Merino sheep. Therefore, it is very necessary to select and breed Hu sheep for growth performance to improve its meat production performance.

[0003] The applicant of the present invention has authorized Chinese invention patents (publication numbers: CN109251986A, CN109182553A, CN109251987A, CN109251985A, CN109182554A, and CN109055579A) that disclose candidate functional genes and SNPs of Hu sheep body size traits screened by GWAS technology. Through population verification of SNPs, SNPs that can be used for molecular marker-assisted breeding have been obtained, 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, CN114480676A), which disclose that there are 15 SNPs in a partial region of intron 1 of the CAPN6 gene. Among them, there are 4 SNPs loci significantly associated with the body weight and body size traits of Hu sheep: 1. g.43756G>A is significantly associated with the body length of Hu sheep. The body length of AA-type individuals is significantly higher than that of GA-type individuals (P<0.05), and there is no significant difference between GG-type individuals and GA and AA-type individuals (P>0.05). 2. g.43815G>A is significantly associated with the body height of Hu sheep. The body height of AA-type individuals is significantly higher than that of GG-type individuals (P<0.05), and there is no significant difference between GA-type individuals and GG and AA-type individuals (P>0.05). 3. g.43851G>A is significantly associated with the body length of Hu sheep. The body length of AA-type individuals is significantly higher than that of GA-type individuals (P<0.05), and there is no significant difference between GG-type individuals and GA and AA-type individuals (P>0.05). 4. g.43917A>G is significantly associated with the body length and daily weight gain of Hu sheep. The body length of AA and GG-type individuals is significantly higher than that of AG-type individuals (P<0.05), and there is no significant difference between AA and GG (P>0.05). The daily weight gain of GG-type individuals from weaning to six months of age is significantly higher than that of AA-type individuals, and there is no significant difference between AG-type individuals and AA and GG.

[0005] In addition, the applicant has also applied for Chinese invention patent applications (CN117757958A, CN117701736A, CN117844943A, CN117683908A), which disclose that there are 4 SNPs in a partial region of intron 2 of the ARHGAP24 gene. Among them, there are 4 SNPs loci significantly associated with the daily weight gain from six months to one year old and / or the one-year-old weight of Hu sheep: 1. g.14979C>T is significantly associated with the daily weight gain from six months to one year old and the one-year-old weight of Hu sheep. The daily weight gain from six months to one year old and the one-year-old weight of TT genotype individuals are significantly higher than those of CT and CC genotypes. 2. g.15042C>T is significantly associated with the daily weight gain from six months to one year old and the one-year-old weight of Hu sheep. The daily weight gain from six months to one year old and the one-year-old weight of TT genotype individuals are significantly higher than those of CT and CC genotypes. 3. g.15255T>C is significantly associated with the daily weight gain from six months to one year old and the one-year-old weight of Hu sheep. The daily weight gain from six months to one year old and the one-year-old weight of CC genotype individuals are significantly higher than those of TC and TT genotype individuals. 4. g.15599G>T is significantly associated with the one-year-old weight of Hu sheep. The one-year-old weight of TT genotype individuals is significantly higher than that of GT and GG individuals.

[0006] AAGAB (Alpha And Gamma Adaptin Binding Protein) is a protein-coding gene. The AAGAB gene consists of 10 exons and corresponding introns, encoding the α / γ-adaptin binding protein p34 (a- and y- adaptin binding protein p34) with 315 amino acid residues. There are two functional domains in the p34 protein encoded by the AAGAB gene. The coding region of the Rab-like GTPase domain located at the N-terminus is in exons 2 to 4, and the coding region of the adaptin binding domain located at the C-terminus is in exons 5 to 10. When AAGAB mutates, the AP2 subunit cannot form the adaptor complex, leading to the degradation of the AP2 subunit and defects in clathrin-mediated endocytosis, ultimately causing diseases. Most studies on this gene have focused on palmoplantar keratoderma (PPK) characterized by abnormal thickening and excessive keratinization of the skin on the palms or feet. Summary of the Invention

[0007] In order to study the correlation between AAGAB gene polymorphisms and the growth traits of Hu sheep and obtain genetic markers related to growth traits, the present invention takes Hu sheep as the research object, uses PCR amplification, direct sequencing of the products, and sequence analysis methods to analyze the polymorphisms of the AAGAB gene, and comprehensively analyzes the correlation between different genotypes of polymorphic sites and different growth traits.

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

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

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

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

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

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

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

[0015] Preferably, the nucleotide sequences of the primer pair are as 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 of each of the upstream and downstream primers, and 10.0 - 12.0 μL of sterilized water.

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

[0018] The present invention takes Hu sheep as the research object, uses PCR amplification, direct sequencing of the product, and sequence analysis methods to analyze the polymorphism of the AAGAB gene, and comprehensively analyzes the correlation between different genotypes of the polymorphic sites and different growth traits. The analysis results show that: g.23443T>C is significantly correlated with the chest circumference of Hu sheep and the daily weight gain from six months old to one year old, and the birth weight, weaning weight, and six - month - old weight of the CC genotype are extremely significantly higher than those of the TT and TC genotype individuals. Description of the Drawings

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

[0020] Figure 2 It is the nucleotide sequence diagram of the amplification product of the present invention. Detailed Embodiments

[0021] Combined with the embodiments of the present invention, the technical solutions in the embodiments will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0022] 1 Materials and Methods 1.1 Sample Collection A total of 133 blood samples of Hu sheep (female sheep) were collected. Blood was collected from the jugular vein, anticoagulated with EDTA, and stored at -20 °C. The sample collection site was Hangzhou Pangda Agricultural Development Co., Ltd. The growth trait indicators measured included: body height, body length, chest circumference, weaning weight, six-month-old weight, and one-year-old weight.

[0023] 1.2 Primer Design According to the sheep AAGAB gene sequence (Acc: 25662) published by Ensembl, PCR amplification primers were designed using DNAMAN 8.0 software. The primer sequences are shown in Table 1. The primers were synthesized by General Biosystems (Anhui) Co., Ltd.

[0024] Table 1 PCR Amplification Primers for Hu Sheep AAGAB Gene

[0025] 1.3 PCR Amplification and Sequence Analysis The PCR reaction system was 25 μL: 12.5 μL of KOD One™ PCR Master Mix - Blue, 0.2 μL of each upstream and downstream primer (10 μM), and 11.1 μL of sterilized water. The PCR reaction conditions were: denaturation at 98 °C for 10 s, annealing at 61 °C for 30 s, extension at 68 °C for 30 s, and 34 cycles. After the PCR was completed, it was stored at 4 °C.

[0026] The amplified products were sent to General Biosystems in Anhui for Sanger sequencing. The Mutation Surveyor 5.02 (Softgenetics, USA) software was used to analyze the sequencing peak maps of each individual to determine the mutation positions and mutation types of each individual.

[0027] 1.4 Data Statistics and Analysis 1.4.1 Polymorphism Information Content Analysis PIC The PIC value was calculated using the little Programe, and the PopGen 32 software was used to calculate the heterozygosity, Shannon information content, gene frequency, and genotype frequency of each SNPs locus.

[0028] Polymorphism information content (PIC) analysis is used to determine and analyze the information content expressed by a genetic marker, representing the probability that an allele obtained by an offspring comes from the same allele of its parent. It is an ideal indicator for measuring the polymorphism of allele fragments. PIC > 0.5 indicates a highly polymorphic locus, 0.25 < PIC < 0.5 indicates a moderately polymorphic locus, PIC < 0.25 indicates a lowly polymorphic locus, and the larger the PIC value, the greater the number of effective alleles and heterozygosity.

[0029] The calculation formula is:

[0030] P i and P j are the allele frequencies of the i-th and j-th alleles respectively; n is the number of alleles.

[0031] The heterozygosity of a locus (He) refers to the average frequency of heterozygous individuals present at each locus. Heterozygosity can objectively reflect the genetic variation level of a population. The larger the average heterozygosity value, the greater the genetic differences within the population, the richer the genetic diversity, the greater the genetic potential, and the better the application effect in animal genetic breeding research; the lower its value, the higher the genetic consistency, indicating less genetic variation within the population and smaller genetic potential. The calculation formula is:

[0032] p i represents the allele frequency of the i-th allele.

[0033] Shannon information content SIC (Shannon information content), the calculation formula is: SIC = -C * logP i where: P i is the frequency of the i-th allele in the population, and C is a constant.

[0034] Gene frequency and genotype frequency ① Genotype frequency = Number of genotype individuals / Number of individuals in the population × 100% ② Gene frequency = Homozygous genotype frequency + 1 / 2 × Heterozygous genotype frequency 1.4.2 Association analysis Use the General Linear Model (GLM; SPSS) to mine SNPs associated with the body weight and body size traits of Hu sheep.

[0035] Since all the individuals analyzed were from the same farm, with the same feeding environment and management conditions, and all were ewes, the farm effect and gender effect were not included in the data modeling.

[0036] The specific model is: Y = Xβ + e Where, Y: the phenotypic value vector of the body size traits and body weight traits of Hu sheep; β: the fixed effect vector such as phenotypic mean, SNP, etc.; e: the residual effect vector; X is the incidence matrix of β.

[0037] 2 Experimental results 2.1 PCR amplification results of SNPs detection of the AAGAB gene in Hu sheep The brightness of the PCR products was high, the bands were single, and there was no non-specific amplification ( Figure 1 ), and the actual PCR products were consistent with the expected PCR amplification products in size, so the subsequent direct sequencing of the PCR products could be carried out.

[0038] 2.2 Analysis of sequencing results The sequencing results were aligned using Mutation Surveyor 5.02 software, and 2 SNPs were found in the AAGAB gene, namely g.23366T>C and g.23443T>C, and both had 3 genotypes, namely TT / TC / CC and TT / TC / CC respectively.

[0039] Table 2 Positions of mutation sites on the amplified sequences

[0040] 2.3 Population genetics analysis of the AAGAB gene As can be seen from Table 3, the effective alleles were 1.07 and 1.48 respectively, the average heterozygosities were 0.07 and 0.32 respectively, and the Shannon information contents were 0.15 and 0.50 respectively. g.23366T>C belonged to low polymorphism (PIC<0.25), and g.23443T>C belonged to moderate polymorphism (0.25<PIC<0.50).

[0041] Table 3 Population genetics analysis of SNP sites of the AAGAB gene

[0042] 2.4 Association analysis between SNPs of the AAGBA gene and growth traits Using the "1.4.2 Association analysis", the association analysis between SNPs of the AAGAB gene and the growth traits of Hu sheep is shown in Table 4. g.23443T>C was significantly correlated with the chest circumference and the weight from six months old to one year old of Hu sheep ( P(<0.05). The chest circumference of individuals with the CC genotype was significantly higher than that of TT genotype individuals. There was no significant difference between the CC genotype and the TC genotype. The daily weight gain from six months to one year old of CC and TC genotypes was significantly higher than that of TT genotype individuals.

[0043] Table 4 Association analysis of AAGAB gene SNPs and growth traits of Hu sheep

[0044] Note: Data with different superscript letters in the same column and the same position were significantly different (P < 0.05).

[0045] 3. Result analysis: 3.1 Correlation analysis of SNP locus g.23366T > C and growth traits of Hu sheep From the perspective of the statistical significance level (P value), the P values corresponding to g.23366T > C in each trait index were all greater than 0.05, indicating that this locus had no significant effect on the growth traits of Hu sheep in this sample population. The differences in indexes such as body height, chest circumference, body length, weaning weight, and weight growth rate among different genotypes (TT, TC, CC) fluctuated little and were not statistically significant. Although individuals with the CC genotype had an absolute advantage in sample size (n = 126), their average weight growth indexes were slightly higher than those of TT / TC genotypes at multiple stages, but the differences were not significant. Therefore, g.23366T > C is not a suitable target locus for molecular marker-assisted breeding.

[0046] 3.2 Correlation analysis of SNP locus g.23443T > C and growth traits of Hu sheep 1) Chest circumference (cm): P = 0.04, reaching a significant difference level. The CC genotype (100.20 ± 6.58) was significantly higher than the TT genotype (94.50 ± 5.78), while the TC genotype (99.07 ± 6.99) was in an intermediate state. The letter annotation (a, b) also indicated that CC > TC > TT, showing a statistical stratification.

[0047] 2.) Daily weight gain from six months to one year old (kg): P = 0.04, also reaching a significant level. Both CC and TC genotypes were significantly higher than the TT genotype (0.17a vs 0.12b kg / day), but there was no significant difference between CC and TC, suggesting that there might be a dominant genetic effect.

[0048] 3) Other trait indexes (body height, body length, birth weight, weaning weight, daily weight gain from weaning to six months old, one-year-old weight, etc.) The P values were all greater than 0.05, indicating no significant statistical differences. However, it is worth noting that the CC genotype was at a relatively superior level in multiple indicators (such as birth weight and body length), having potential reference value.

[0049] 3.3 Summary of the Application Value of Molecular Breeding The locus g.23443T>C is the only SNP significantly associated with growth traits (chest circumference and growth rate) in the present invention. Individuals with the CC / TC genotypes showed better growth potential. This locus has moderate polymorphism (PIC = 0.50), possessing the effectiveness for population screening and can be used as a molecular discriminant marker for the core breeding population. It can be used to develop commercial molecular breeding tools, such as SNP detection kits, to guide early identification of high-quality breeding sheep in farms.

[0050] The above is the description of the embodiments of the present invention. Through the above description of the disclosed embodiments, those skilled in the art can implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. 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 these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel points disclosed herein.

Claims

1. A reagent for detecting molecular markers significantly associated with the chest circumference and daily weight gain from six months to one year old of Hu sheep, characterized in that, The molecular marker is located at g.23443T>C, which is at the 264th site of SEQ ID NO:1 and is either 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 to one year old of individuals with the CC and TC genotypes is significantly higher than that of individuals with the TT genotype.

2. A primer pair for identifying the molecular marker described in claim 1.

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

3.

4. A kit comprising the reagent described in claim 1 or the primer pair described in any one of claims 2-3.

5. Use of the reagent described in claim 1 or the primer pair described in any one of claims 2-3 or the kit described in claim 4 in the auxiliary breeding for screening the chest circumference and the daily weight gain from six months to one year old of Hu sheep.

6. Use of an amplification product in the auxiliary breeding for screening the chest circumference and the daily weight gain from six months to one year old of Hu sheep, wherein the amplification product is obtained by amplification using the reagent described in claim 3 and the nucleotide sequence is as shown in SEQ ID NO:

1.

7. A method for screening chest circumference and weight gain traits from six months to one year old, characterized in that, The method comprises the following steps: extracting the genomic DNA of Hu sheep, performing PCR amplification using the primer pair, detecting the molecular marker in the amplification product, so as to screen the traits of the chest circumference and the daily weight gain from six months to one year old of Hu sheep; the molecular marker is located at g.23443T>C, which is at the 264th site of SEQ ID NO:1 and is either 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 to one year old of individuals with the CC and TC genotypes is significantly higher than that of individuals with the TT genotype.

8. The method according to claim 7, wherein The nucleotide sequences of the described primer pair are as shown in SEQ ID NO:2 and SEQ ID NO:

3.

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

10. The method according to claim 8 or 9, characterized in that, The PCR reaction program is: denaturation at 95 - 98 °C for 8 - 15 s, annealing at 60 - 65 °C for 25 - 35 s, extension at 65 - 70 °C for 25 - 35 s, reacting for 30 - 40 cycles, and storing at 0 - 5 °C after the PCR is completed.

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