CN102643914A - Method for detecting depression sheep fecundity - Google Patents

Abstract

The invention discloses a method for predicting the fecundity of Wadi sheep by using the Fec ^B gene, which is to detect the 746th nucleotide of the Fec ^B gene in the Wadi sheep genome to be tested, thereby determining the genotype of the Wadi sheep, and then determining the Wadi sheep through the genotype Sheep fecundity; if the 746th nucleotide of the Fec ^B gene in the Wadi sheep genome is A, the genotype of its homozygote is ++, and the 746th nucleotide of the Fec ^B gene in the Wadi sheep genome is When G, the genotype of the homozygote is BB; the genotype of the heterozygote is B+; the fecundity of the BB genotype is higher than that of the B+ genotype, and the fecundity of the B+ genotype is higher than that of the ++ genotype. The invention utilizes PCR-SSCP and PCR-RFLP genetic marker polymorphism to determine the fecundity of Wadi sheep, can quickly and easily detect the fecundity of Wadi sheep, and provides an accurate and convenient method for detecting the fecundity of Wadi sheep for breeding of Wadi sheep method.

Description

A method for testing the fecundity of Wadi sheep

technical field

A method for testing the fecundity of lowland sheep.

Background technique

The Fec ^B gene was discovered in the Booroola Merino sheep breed in Australia in 1985, and is the first major gene described as affecting the ovulation rate and fecundity of sheep. Booroola mutations, point mutations in the coding region of the BMPR-IB locus, have been crossed into other sheep breeds, and other productive sheep breeds other than Booroola Merino sheep have also been found in the natural state. The Fec ^B gene is located in the narrow region of sheep chromosome 6 6q23~q31, between osteopontin and epidermal growth factor. Genotype assignment at the Fec ^B locus, initially based on detection of ovulation rates and lambing records, later used to link genetic markers to the Booroola gene, can now be obtained by molecular genotyping of individuals at the BMPR-IB locus . One copy of Fec ^B increases the number of ovulations by 1.3-1.6, two copies of FecB increases the number of ovulations by 2.7-3.0, the fecundity of ewes carrying one copy of Fec ^B increases by 0.9-1.2, and the ewes carrying two copies of Fec ^B The fecundity of ewes increased by 1.1 to 1.7 (Chu Mingming, Di Ran, Ye Sucheng, Fang Li, Liu Zhonghui, Peng Zhilan, Zhang Hexi, Jia Lihua, Sun Jie, Feng Tao. Molecular detection method of Fec ^B , the main gene for multiple births in sheep Establishment and application [J]. Journal of Agricultural Biotechnology, 2009, 17(1): 52-58).

Lowland sheep (abbreviated as lowland sheep) is an excellent local sheep breed distributed in the coastal areas of the saline-alkaline depressions in northern Shandong. After textual research, the lowland sheep is a unique and excellent local breed gradually formed in the coastal area after more than 400 years of domestication, adaptation, and breeding since the Ming Dynasty. Lowland sheep can be in oestrus all year round, especially in spring and autumn. The lambing rate of the first litter is not significantly different from that of the Small-tailed Han sheep, which belongs to the multiparity sheep breed. Lowland sheep are resistant to rough feeding and grow faster. In terms of body shape, lowland sheep have no horns, short and strong limbs, square tails, and a docile and non-aggressive personality, which makes lowland sheep have a high slaughter rate and rapid growth and development. After long-term selection and breeding, the lowland sheep have adapted to the local barren pastures, and the perennial grazing has exercised the quality of the flock. In particular, the local coastal wetlands are severely saline-alkali, which is very corrosive to sheep's feet, and it is difficult for other breeds of sheep to adapt. The lowland sheep are well adapted to the local environment and have the ability to resist hoof rot.

Montgomery et al. (1993) found for the first time that the Fec ^B gene was closely linked to the microsatellite markers OarAE101 and OarHH55, with a distance of 13 cM and 20 cM, respectively, and these two genes were in turn linked to the secreted phosphoprotein 1 gene located on the human chromosome HSA4q11-q12 ( An RFLP marker linkage of SSP1). Subsequently, the research group used linkage analysis to study Booroola sheep half-sib families and 17 full-sib families, and found that platelet-derived growth factor receptor-alpha gene (PDGFRA) and αS1 casein gene (alpha sl-caseingene, CSN1S1) and microsatellite markers BM143 and OarHH55 were linked, and the genetic distances were 12cM, 29cM, and 33cM, respectively. When Lord et al. used microsatellite OarAE101 and BM1329 as markers to study the Fec ^B gene, they found that the Fec ^B gene was present on the 97bp allele at the OarAE101 site and the 162bp allele at the BM1329 site, so the Fec ^B gene was further positioned at In the 10cM linkage group between BM1329 and OarAE101 on chromosome 6 (Montgomery G W, et al. Nat Genet, 1993, 4(4):410-414).

Further research by Montgomory found that the Fec ^B gene is linked with the EGF (epidermal growth factor) gene at a distance of 26cM, and EGF is located on 4q25 of the human chromosome, which is an important step towards the cloning of the Fec ^B mutation position (Montgomery G W, et al. Genomics, 1994, 22(1): 148-153). Wilson et al. (2001) further located the Fec ^B gene on sheep chromosome 6 6q23~q31 on the basis of previous studies, which is syntenic with human chromosome 4q21~q25. It is also reported that the gene on chromosome 6 of sheep is located on chromosome 8 of pig, and a site (Wilson T, etal.Biol Reprod, 2001, 64 (4) that improves ovulation rate has been found on chromosome 8 : 1225-1235).

Wilson et al., Souza et al., and Mulsant et al. (2001) discovered almost simultaneously that the bone morphogenetic protein receptor IB gene (BMPR-IB) on human chromosome 4 q22-q23 was syntenic to the region of sheep chromosome 6 where the Fec ^B gene was located. ) correlated with the ovulation rate of Booroola Merino sheep.

(2008) by Elisha Gootwine etc. (2008) by the ++ of Fec ^B site and the investigation of B+ genotype fecundity, the result shows, B site has multiplicative effect on fecundity, because the fecundity of B+ genotype is significantly high Fecundity of ++ genotype (P<0.05) (Elisha Gootwine, Shay Reicher, Alexander Rozov. Prolificacy and lamb survival at birth in Awassi and Assaf sheepcarrying the FecB(Booroola) mutation. [J]. Animal Reproduction Science, 2008, 108(2):402-411).

A.K.Mishra et al. (2009) analyzed the fecundity of 51 hybrid sheep of Garole and Malpura. The results showed that the cumulative litter size (CLS), the cumulative number of weaned lambs (CWL) and the cumulative reproductive efficiency of ewes (CEPE) On hybrid ewes, the B+ genotype is relatively higher than the ++ genotype ewes (A.K.Mishra, A.L.Arora, S.Kumar, L.L.L.Prince. Studies on effect of Booroola(FecB) genotype on lifetime ewes productivity efficiency, littersize and number of weaned lambs in Garole×Malpura sheep [J]. Animal Reproduction Science, 2009, 113(3): 293-298);

S.Kumar et al. (2008) found the Fec ^B mutation in Kendrapada sheep through the experimental method of PCR-RFLP. In 46 Kendrapada sheep individuals, 26 BB genotypes, 15 B+ genotypes, and 5 ++ genotypes , the genotype frequency of B was about 0.73, and the results showed that despite the high mutation frequency of Fec ^B , the gene was not fixed in the population as reported in Garole sheep (S. Kumar, AK Mishra, APKolte, SKDash, SA Karim. Screening for Booroola (FecB) and Galway (FecXG) mutations in Indian sheep [J]. Small Ruminant Research, 2008, 80(4): 57-61).

Wang Genlin et al. (2003) detected the DNA samples of Hu sheep, Small-tailed Han sheep, and Xinjiang fine-wool sheep by using restriction enzyme polymorphic PCR technology, and found that there were Fec ^B multiple birth genes in Hu sheep and small-tailed Han sheep, while Xinjiang fine-haired sheep None of the sheep carry the Booroola gene (Wang Genlin, Mao Xinzhi, George H Davis, Zhao Zongsheng, Zhang Lijun, Zeng Yongqing. DNA analysis found that the Booroola (Fec ^B ) gene exists in Hu sheep and small-tailed Han sheep in China [J]. Journal of Nanjing Agricultural University, 2003, 26(1):104-106).

Zhang Liping (2004) made a relatively comprehensive analysis on the discovery, location, association and application of the Fec ^B gene, and showed that the mutation of the BMPR-IB gene is completely consistent with the behavior of the Fec ^B gene, proving that the BMPR-IB gene controls the behavior of the Booroola Merino sheep. The main gene of high fecundity traits (Zhang Liping. Research progress of Booroola sheep multifetal gene Fec ^B [A]. 2003-2004 National Symposium on Sheep Production and Academic Proceedings, 2004.).

Liu Nan et al. (2010) proved that in the hybrid population of Small-tailed Han sheep and Suffolk and Dorper sheep, the multifetal gene Fec ^B has a significant polymorphism detection effect, and can be used as an effective candidate gene for breeding new breeds of meat sheep (Liu Nan, Liu et al., 2010). Nan, Liu Xiaoxiao, Liu Meng, Qu Xuxian, Wang Jinwen, Li Jing. Fec ^B and BMP15 genes as candidate genes for meat sheep breeding[J]. Heilongjiang Animal Husbandry and Veterinary Medicine, January 2010 (Part 1): 39-40) .

Cui Xukui et al. (2010) used the Fec ^B molecular detection technology of sheep multifetal gene to detect and analyze the genotype of Duhan hybrid mutton sheep and its influence on fecundity and lamb growth and development. It was found that the Fec ^B gene is not only the main gene affecting the fecundity of Duhan hybrid mutton sheep, but also affects the growth and development of lambs within a certain ^period of time (Cui Xukui, Wang Jinwen, Wang Deqin, Zhang Guoping, Wang Ke, Huang Qinghua, Meng Xianfeng. ×The influence of cold hybrid mutton sheep on fecundity and lamb growth and development [J]. Shandong Agricultural Science, 2010, 8: 98-100).

Wang Jinwen et al. (2010) proved that the Fec ^B gene may be the main gene controlling the multi-height traits of Luxi mutton sheep. , Meng Xianfeng. Using Fec ^B gene to breed Luxi mutton sheep with multiple births [J]. Journal of Livestock Ecology, 2010, 31(5): 23-25).

Li Fenge (2010) made a comprehensive review on the proposal of Fec ^B gene, genotype determination, physiological effects and isolation and cloning (Li Fenge, Xiong Yuan. Research progress on isolation and cloning of sheep Fec ^B gene[J].Animal Science Abroad , 2010, 28(6): 42-44).

Ren Yanling et al. (2011) studied the multi-lamb mechanism of Wadi sheep from the molecular level, and analyzed the relationship between Fec ^B gene polymorphism and fecundity in Wadi sheep by using PCR-RFLP method. After statistical analysis, BB and B+ genotypes in Wadi sheep The fecundity of sheep is significantly higher than that of the ++ genotype, indicating that the Fec ^B gene may be one of the main genes for multiple lamb performance in Wadi sheep (Ren Yanling, Shen Zhiqiang, Li Min et al. Relationship between Fec ^B gene polymorphism and fecundity of Wadi sheep Research. Chinese Animal Husbandry and Veterinary Medicine, 2011, 38(7): 159-162).

Many scientists have also conducted a lot of analysis on the polymorphism and linkage between sheep microsatellite gene and Fec ^B gene. The research results of Zhang Baoyun et al. (2009) showed that the microsatellite locus 300U can only reflect the Fec ^B locus information to a certain extent (Zhang Baoyun, Chu Mingxing, Wang Pingqing, Fang Li, Di Ran. The number of sheep microsatellite 300U and Fec ^B genes State and linkage analysis [J]. Journal of China Agricultural University, 2009, 14(5): 86-92). (2009) showed that linkage disequilibrium analysis showed that there was a certain linkage disequilibrium between the B allele of the Fec ^B gene of Small Tail Han sheep and the 100bp allele of the BMS2508 microsatellite locus, while the + allele was associated with the BMS2508 microsatellite locus. The 110bp and 114bp alleles of the satellite loci are in certain linkage disequilibrium (Li Yanlu, Chu Mingming, Chen Hongquan, Fang Li, Di Ran, Ma Yuehui, Li Kui. Polymorphism and linkage analysis of sheep microsatellite BMS2508 and Fec ^B genes[J ]. Heredity, 2009, 31(5): 500-507). (2009) showed that there is a strong linkage relationship between the 182bp allele of the OarJL36 microsatellite locus and the B allele of the Fec ^B gene of Small-tailed Han sheep, which is closely related to the main effect gene of multiple lambs in Small-tailed Han sheep. A genetic marker of linkage (Chu Mingxing, Zhang Baoyun, Wang Pingqing, Fang Li, Di Ran, Ma Yuehui, Li Kui. Polymorphism and linkage analysis of sheep microsatellite OarJL36 and Fec ^B genes [J]. Chinese Agricultural Sciences, 2009, 42(6):2133-2141). Research by Zhang Lin et al. (2009) showed that there is a certain linkage disequilibrium relationship between the 98bp allele of LSCV043 microsatellite locus and the B allele of the Fec ^B gene of Small-tailed Han sheep, which is the main effect gene for multiple lambs in Small-tailed Han sheep. A closely linked genetic marker (Zhang Lin, Li Chunmiao, Chu Mingming, Chen Hongquan, Li Xuewei, Fang Li, Di Ran, Ma Yuehui, Li Kui. Linkage analysis of microsatellite locus LSCV043 and Fec ^B gene in Small Tail Han sheep[J].Agrobiology Technical Journal, 2009, 17(4): 621-628).

According to the records of "Chinese Sheep Breeds" ("China Sheep Breeds" compilation group, 1989), the average number of live lambs of lowland sheep is 2.80, which is a breed of multiparous sheep in my country.

Contents of the invention

The purpose of the present invention is to provide a method for detecting the fecundity of swale sheep.

The method for detecting the fecundity of Wadi sheep provided by the present invention is to detect whether the 746th nucleotide of the Fec ^B gene in the Wadi sheep genome to be tested is A or mutated to G, thereby determining the genotype of the Wadi sheep, and then passing the gene type to determine the fecundity of Wadi sheep; the nucleotide sequence GENBANK ACCESSIONNUMBER of the Fec ^B gene is the 156bp to 3255bp nucleotide of AF357007;

The method for determining the genotype of Wadi sheep is: if the 746th nucleotide of the Fec ^B gene in the Wadi sheep genome is A, the genotype of its homozygote is ++; the Fec ^B gene in the Wadi sheep genome When the 746th nucleotide is G, their homozygous genotype is BB; their heterozygous genotype is B+;

The method for determining the fecundity of the swale sheep by genotype is as follows: the fecundity of the swale sheep of the BB genotype is higher than that of the swale sheep of the B+ genotype, and the fecundity of the swale sheep of the B+ genotype is higher than that of the ++ genotype swale sheep.

There are two methods for detecting whether the 746th nucleotide of the FecB gene in the Wadi sheep genome is A or a mutation G. The first method is to use PCR to amplify the nucleotide sequence GENBANK ACCESSION NUMBER in the Wadi sheep genome. The 626bp to 813 nucleotide fragment of AF357007, and the amplified product was detected by SSCP, and two electrophoresis bands were obtained, the distance between the two bands was the farthest, and the thinner band was the ++ genotype. The two bands are the closest and the thinner bands are the BB genotype, and the two bands are thicker and the distance between the first two is the B+ genotype (as shown in Figure 2); the second is to use the PCR method Amplify the nucleotide sequence GENBANK ACCESSION NUMBER in the Wadi sheep genome from the 638bp to 778th nucleotide fragment of AF357007, and perform RFLP detection on the amplified product. If two bands can appear, and they are located at 140bp and 110bp, it is B+ genotype, if a band can appear, and it is located at 140bp, it is ++ genotype, if a band can appear, and it is located at 110bp, it is BB genotype (as shown in Figure 3).

In the method, the amplification primer pair of the PCR method has the nucleotides described in the sequences 1, 2, 3 and 4 in the sequence listing.

In the method, the fecundity is the number of litters per litter.

The method of the present invention adopts single strand conformation polymorphism (single strand conformation polymorphism, SSCP) and restriction endonuclease fragment polymorphism (restriction fragmentlength polymorphism, RFLP) method to carry out SNP in Fec ^B gene ( singlenucleotide polymorphism (SNP) detection to compare the polymorphisms of the Fec ^B gene in Wadi sheep breeds, and to conduct a comparative analysis of the DNA fragments with SSCP and RFLP polymorphisms, and to find out the factors related to fecundity (fecundity) Genetic markers, statistical results show that polymorphisms of the genetic markers can determine the fertility of Wadi sheep. The method of the present invention uses the genetic markers to establish SSCP and RFLP to detect the genotype of Wadi sheep, and utilizes the genotype to determine its effective method for fecundity. Experiments have proved that the method can quickly and easily detect the fecundity of Wadi sheep . The method of the invention provides an accurate and convenient method for detecting the fecundity of sheep molecular breeding.

Description of drawings

Fig. 1 is the PCR product of two pairs of primers (P1, P2) of Fec ^B gene.

Figure 2 is the SSCP analysis of primer P1 on the amplified fragment of Wadi sheep.

Figure 3 is the RFLP analysis of primer P2 on the amplified fragment of Wadi sheep.

Detailed ways

The experimental methods mentioned in the following examples are conventional methods unless otherwise specified.

The main reagents used in the following examples 10PCR Buffer, dNTP, Taq DNA polymerase, DNA MakerpBR322/Hae III, AvaII restriction endonuclease PCR primers, etc. were purchased from Shanghai Bioengineering Company.

Example 1. Establishment of a method for detecting the fecundity of Wadi sheep and its effect verification

1. The establishment of a method for detecting the fecundity of lowland sheep

1) template material preparation

The ear tissue samples of Wadi sheep were collected and the fecundity and parity were recorded. 98 Wadi sheep were collected from Binzhou, Shandong. Genomic DNA of sheep was extracted by phenol-chloroform method, dissolved and diluted in sterilized TE and stored at -20°C for later use.

2) Primer design

According to the complete coding sequence of the sheep Fec ^B gene (AF357007) reported by GenBank, two pairs of primers were designed at the coding region sequence (GENBANK ACCESSION NUMBER is the 156bp to 3255bp nucleotides of AF357007) with Oligo 7.0 software. Synthesized by Gongbio Technology Co., Ltd. Primer sequences and amplified fragment sizes are as follows:

Primer 1 was used for PCR-SSCP detection, F: 5'-AGATTGGAAAAGGTCGCTATG-3' (SEQ ID No.1); R: 5'-ACCCTGAACATCGCTAATACA-3' (SEQ ID No.2), amplified 187bp fragment (from GENBANK The accession number is the 626bp to 812th nucleotide sequence of AF357007, see SEQ ID No.5).

Primer 2 is used for PCR-RFLP detection, F: 5'-GTCGCTATGGGGAAGTTTGGATG-3' (SEQ ID No.3); R: 5'-CAAGATGTTTTCATGCCTCATCAACACGGTC-3' (SEQ ID No.4), amplifies the fragment of 140bp (from GENBANK The accession number is the 638bp to 777th nucleotide sequence of AF357007, see SEQ ID No.6).

Using the genomic gene extracted from the Wadi sheep experimental material obtained in step 1 as a template, the above primers were used for PCR amplification.

The PCR amplification system is as follows: the PCR amplification system of primer 1 and primer 2 is the same. The total volume of the PCR amplification system is 20 μL, including 2.5 μL of 10×Buffer (containing Mg ²⁺ ), 2 μL of 10 mmol/LdNTP, 1.0 μL of 10 μmol/L upstream and downstream primers, 1 μL of 100 ng/μL DNA template, 0.3 μL of rTaq enzyme, and 12.2 μL of ionized water.

PCR amplification conditions were as follows: Primer 1 was pre-denatured at 94°C for 5 min, denatured at 94°C for 30 s, annealed at 55.2°C for 30 s, extended at 72°C for 30 s, 32 cycles, extended at 72°C for 10 min, and stored at 4°C. Primer 2 was pre-denatured at 94°C for 5 min, denatured at 94°C for 30 s, annealed at 64.6°C for 30 s, extended at 72°C for 30 s, 31 cycles, extended at 72°C for 10 min, and stored at 4°C.

The PCR products obtained by amplifying the Wadi sheep genome with the two pairs of primers designed were detected by polyacrylamide gel electrophoresis. PCR product detection: 5 μL of amplification products plus 2 μL of buffer (0.05% xylene blue; 0.05% bromine Phenol blue; 0.02mol/L EDTA, add formamide to 10ml), the amount of DNA Marker is 3μL. 120V at room temperature for 2.5h, silver staining. The results showed that the amplified fragment was consistent with the target fragment in size and had good specificity (Figure 1), and could be directly analyzed by SSCP and RFLP. Lanes 1-6 in Figure 1 are the electrophoresis images of the fragments amplified by primer 2, and lanes 7-11 are the electrophoresis images of the fragments amplified by primer 1; lane M in Figure 1 is pBR322marker.

3) SSCP analysis and RFLP analysis

SSCP analysis

The PCR products amplified by the two pairs of primers obtained in step 1) were respectively subjected to SSCP analysis in Wadi sheep, and the specific method was as follows: draw 10 μL of PCR amplification products and mix them with 10 μL of denaturing buffer; , denatured at 98°C for 10 minutes, took it out and placed it in an ice box and placed it in a refrigerator at -25°C for 5 minutes to complete the denaturation; cut off the power supply, put the denatured mixed solution into each gel hole with a 10μL pipette, turn on the power supply, and use 220V voltage After 15 minutes, the two bands of bromophenol blue and xylene cyanol were observed to separate, and then electrophoresed at a constant voltage of 120V for 10 hours, and the color was developed by silver staining. Images were taken and analyzed with AlphaImager ^™ 2200 and 1220 Documentation and Analysis Systems (Alpha Innotech Corporation, San Leandro, CA, USA).

It was found that there were three genotypes in the fragment amplified by primer 1, which were named BB, B+, and ++ (Fig. 2), that is, two bands were obtained by electrophoresis of the non-denatured polyacrylamide of the amplified product. The distance between the two bands is the farthest, and the thinner band is the ++ genotype (lane 1, 2, 3 in Figure 2), the two bands are the closest, and the thinner band is the BB genotype (Figure 2 Swimming lanes 7, 8, 9), the two bands are thicker, and the distance between them is B+ genotype (swimming lanes 4, 5, 6 in Figure 2). In Figure 2, lanes 1, 2, and 3 are ++ genotype; lanes 4, 5, and 6 are B+ genotype; lanes 7, 8, and 9 are BB genotype.

RFLP analysis

Respectively carry out RFLP analysis to the PCR product amplified in Wadi sheep by two pairs of primers that step 1) obtains respectively, and specific method is as follows:

PCR-RFLP digestion system: the PCR amplification product of primer 2 was digested with AvaII restriction endonuclease. The total volume of the digestion reaction was 15 μL. Among them, 5 μL of PCR product, 1.0 μL of AvaII restriction endonuclease, 1.5 μL of 10×Buffer, and 7.5 μL of deionized water. Shake and centrifuge at 7500r/min for 30s, and the enzyme digestion reaction condition is 37°C water bath for 4h.

Detection of PCR-RFLP products: 5 μL digested product plus 2 μL loading buffer (0.05% xylene blue; 0.05% bromophenol blue; 0.02mol/L EDTA, add formamide to 10 ml), the amount of DNA Marker is 3 μL. 120V at room temperature for 3.5h, silver staining. Images were taken and analyzed with AlphaImager ^™ 2200 and 1220 Documentation and Analysis Systems (Alpha Innotech Corporation, San Leandro, CA, USA).

As a result, it was found that there were three genotypes in the amplified fragment of primer 2, which were named BB, B+, and ++ (Figure 3). 1, 2, 5, 10, 12-16, 18-19), if a band can appear, and it is located at 140bp, it is ++ genotype (lane 6, 8, 9 in Figure 3), if a band can appear Band, and located at 110bp, is the BB genotype (lanes 3, 4, 7, 17 in Figure 3). In Figure 3, lanes 6, 8, and 9 are ++ genotypes; lanes 1, 2, 5, 10, 12-16, and 18-19 are B+ genotypes; lanes 3, 4, 7, and 17 are BB genotypes.

The experimental results show that the above two methods can judge the type accurately, have good stability and reliability, and the judgment results are consistent. According to the analysis results of PCR-SSCP and PCR-RFLP, samples of different genotypes were selected for sequence determination. After the PCR products were identified by electrophoresis, they were sequenced by the AB IPRISM 377 DNA automatic sequencer of Shanghai Bioengineering Technology Service Co., Ltd.

2. Statistical analysis of Fec ^B genotype detection and its relationship with fecundity

According to the method of step 2), the Fec ^B genotype of Wadi sheep was detected with primer 1 and primer 2, and the genotype frequency and allele frequency of Wadi sheep were calculated. The statistical results are shown in Table 1.

Table 1 Allele frequency and genotype frequency of FecB locus in Wadi sheep

Note: Numbers in parentheses are sample sizes.

The least squares analysis was used for the association analysis between different genotypes of sheep and lambing traits, and the following generalized linear model (GLM) was constructed:

Yi _jk ＝μ+G _j +I _k +e _ijk

(Y _ijk is the observed value of traits; μ is the population mean; G _j is the genotype effect; I _k is the family effect; e _ijk is the random residual effect), using SPSS biological analysis software to analyze the correlation between genotype and swale sheep fertility sex. Table 2 shows the least square mean and standard error of the fecundity of lowland sheep of different genotypes.

Table 2 Least squares mean and standard error of different genotypes and lowland sheep fecundity

Note: The same uppercase letter indicates no significant difference (p>0.05); different lowercase letters indicate significant difference (0.01<p<0.05); different uppercase letters indicate extremely significant difference (p<0.01).

The results in Table 2 show that the fecundity of the BB genotype is 0.90837 higher than that of the ++ genotype, which indicates that the BB and ++ genotypes have certain effects on the lambing traits of Wadi sheep, and the different genotypes at this locus are in The difference in fecundity reached a very significant level (P<0.01); the fecundity of B+ genotype was 0.49581 higher than that of ++ genotype, which indicated that B+ and ++ genotypes had a certain influence on the lambing traits of Wadi sheep, and The difference in fecundity of different genotypes at this locus did not reach a significant level (P>0.05); The fecundity of BB genotype is 0.41256 higher than that of B+ genotype, which indicates that BB and B+ genotypes have a certain influence on the lambing traits of Wadi sheep, and the difference in fecundity of different genotypes at this locus is extremely significant (P<0.01). This result indicated that the polymorphisms of Wadi sheep could be detected by using the above-mentioned primers 1 and 2 genotype polymorphisms.

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Claims (6)

1. A method for detecting the fecundity of Wadi sheep is to detect whether there is an A → G mutation at the 746bp of the Fec ^B gene coding region in the Wadi sheep genome to determine the genotype of the Wadi sheep, and then pass the genotype Determine the fecundity of Wadi sheep; the nucleotide sequence GENBANK ACCESSION NUMBER of the FecB gene is the 156bp to 3255bp nucleotides of AF357007; The method for determining the genotype of Wadi sheep is: if the 746th nucleotide of the Fec ^B gene in the Wadi sheep genome is A, the genotype of its homozygote is ++; the Fec ^B gene in the Wadi sheep genome When the 746th nucleotide is G, their homozygous genotype is BB; their heterozygous genotype is B+; The method for determining the fecundity of the swale sheep by genotype is as follows: the fecundity of the swale sheep of the BB genotype is higher than that of the swale sheep of the B+ genotype, and the fecundity of the swale sheep of the B+ genotype is higher than that of the ++ genotype swale sheep. 2. The method according to claim 1, characterized in that: the method for detecting whether the 746th nucleotide of the FecB gene in the Wadi sheep genome is A or mutation G is to utilize the PCR method to amplify the FecB gene in the Wadi sheep genome. The nucleotide sequence GENBANK ACCESSION NUMBER is a fragment from 626bp to 812 nucleotides of AF357007, and the amplified product was detected by SSCP, and two electrophoresis bands were obtained, and the distance between the two bands was the farthest. The thinner one is the ++ genotype, the two bands closest to each other, and the thinner band is the BB genotype, the two thicker bands, and the distance between the first two bands is the B+ genotype. 3. The method according to claim 2, characterized in that: the amplification primer pair of the PCR method has the nucleotides described in sequences 1 and 2 in the sequence listing. 4. The method according to claim 1, characterized in that: the method for detecting whether the 746th nucleotide of the FecB gene in the Wadi sheep genome is A or mutation G is to utilize the PCR method to amplify the FecB gene in the Wadi sheep genome. The nucleotide sequence GENBANK ACCESSION NUMBER is a fragment of the 638bp to 777th nucleotide of AF357007, and the amplified product is detected by RFLP. If two bands appear, and they are located at 140bp and 110bp respectively, it is a B+ genotype. If a band appears at 140bp, it is ++ genotype; if a band appears at 110bp, it is BB genotype (as shown in Figure 3). 5. The method according to claim 2, characterized in that: the amplification primer pair of the PCR method has the nucleotides described in sequence 3 and 4 in the sequence listing. 6. according to the method described in any one in claim 1-4, it is characterized in that: described fecundity is the number of lambs per tire.

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