A SNP molecular marker related to sheep litter size and its application

Abstract

The invention discloses a SNP molecular marker related to the lambing number trait of sheep and its application, belongs to the field of genetic engineering, detects the genotype of the 29907419th site or / and the 29925629th site of chromosome 6 of the sample sheep, and the reference genome version is ARS-UI_Ramb_v2.0. The genotypes of the two sites are used for prediction: when the genotype of the 29907419th site is GG or GA, the number of lambs and the number of live lambs are higher, and the number of lambs and the number of live lambs of the GG genotype are higher than those of GA, and when the genotype is AA, the number of lambs and the number of live lambs are lower; when the genotype of the 29925629th site is TT or TC, the number of lambs and the number of live lambs are higher, and the number of lambs and the number of live lambs of the TT genotype are higher than those of TC, and when the genotype is CC, the number of lambs and the number of live lambs are lower. Provide new marker resources for marker-assisted selection of the lambing number trait of sheep.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a SNP molecular marker related to the lamb number trait of sheep and an application thereof. Background Art

[0002] Sheep are one of the important sources of animal protein for humans (Goldansaz et al., 2022). Increasing the number of lambs born in sheep has huge economic benefits for sheep farming. Due to the low heritability of litter size in sheep, traditional breeding methods are difficult to quickly increase litter size. Therefore, exploring candidate genes and their various genetic variations that affect litter size in sheep, and using these variations in molecular marker-assisted selection (MAS) breeding of sheep to increase litter size and improve the production efficiency of large-scale farming is a common research method at present (Abd El-Hack et al., 2018).

[0003] UNC5C (UNC-5netrin receptor C) is a member of the UNC-5 family of netrin receptors. Current studies have shown that UNC5C is closely related to neural development and acts as a pro-apoptotic molecule that synergizes with its ligand netrin-1 to control axonal migration.

[0004] UNC5C has been widely studied in the field of human diseases, mainly in the field of tumor cancer and Alzheimer's disease. In recent years, it has been found that UNC5C is associated with reproductive traits such as polycystic ovary syndrome. In animals, the research results of UNC5C are very limited. It has been reported that UNC5C mutant male mice have reduced germ cell apoptosis; in Holstein cows, UNC5C is related to the conception rate of embryonic development, which may affect the development of embryos before implantation; in addition, this gene is also related to genes in the cell apoptosis pathway and endometriosis. There are no reports of this gene being associated with reproductive traits in sheep. In our previous studies on sheep, we found that UNC5C is significantly highly expressed in sheep brain tissue and gonadal tissues such as ovaries and testes, and its subcellular localization is mainly in the plasma membrane and nucleus. Summary of the invention

[0005] The technical problem to be solved by the present invention is: how to provide a SNP molecular marker related to the sheep lamb number trait and provide a new molecular marker site for the determination of the sheep lamb number trait.

[0006] The technical solution of the present invention is: a method for predicting the number of lambs born in sheep, detecting the genotype of the 29907419th site or / and the 29925629th site of chromosome 6 of the sample sheep, the reference genome version is ARS-UI_Ramb_v2.0, and prediction is made through the genotypes of these two sites:

[0007] When the genotype of the 29907419th site is GG or GA, the number of lambs born and the number of lambs born alive are higher, and the number of lambs born and the number of lambs born alive of the GG genotype are higher than those of the GA, and when the genotype is AA, the number of lambs born and the number of lambs born alive are lower;

[0008] When the genotype of the 29925629th locus is TT or TC, the number of lambs born and the number of lambs born alive are higher, and the number of lambs born and the number of lambs born alive of the TT genotype are higher than those of the TC. When the genotype is CC, the number of lambs born and the number of lambs born alive are lower.

[0009] The invention discloses an application of a material for detecting the genotype of a SNP site in the prediction of lambing number traits of sheep or in assisting breeding. The SNP sites have two locations on chromosome 6 of sheep, one of which is located at position 149 of the nucleotide sequence shown in SEQ ID No.1 and has a G / A polymorphism, and the other SNP is located at position 188 of the nucleotide sequence shown in SEQ ID No.2 and has a T / C polymorphism.

[0010] Furthermore, the substance has two pairs of primers, wherein the nucleotide sequence of one pair of primers is shown as SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequence of the other pair of primers is shown as SEQ ID No.5 and SEQ ID No.6.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention discloses that the sheep UNC5C gene is associated with the sheep lamb number trait, and finds that two SNP sites in the UNC5C gene can be used as molecular markers for molecular marker screening of the lamb number trait, laying a foundation for further studying the molecular mechanism of the UNC5C gene on the sheep lamb number trait, and providing a new marker resource for marker-assisted selection of the sheep lamb number trait. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Manhattan plots were analyzed using selection signals from different sheep breeds, and a significant segment was mapped to chromosome 6, containing the UNC5C gene.

[0014] Figure 2 Genome-wide association analysis of litter size phenotypes showed that the significant locus segment included the UNC5C gene.

[0015] Figure 3 Phenotypic association analysis of the number of lambs born in consecutive multiple births in Hu sheep ewes showed that the significant locus segment included the UNC5C gene.

[0016] Figure 4Relationship diagram between Chr 6_29907419 locus genotype and the traits of lambing number and number of lambs born alive.

[0017] Figure 5 Relationship diagram between Chr 6_29907419 locus genotype and the traits of lambing number and number of lambs born alive.

[0018] Figure 6 Gel electrophoresis of the amplified product of Chr 6_29907419 site.

[0019] Figure 7 Sequencing results of three genotypes at Chr 6_29907419 locus (the sequence shown is the reverse complementary sequence).

[0020] Figure 8 Gel electrophoresis of the amplified product of Chr 6_29925629 site.

[0021] Fig. 9 Sequencing results of three genotypes at Chr 6_29925629 locus. DETAILED DESCRIPTION

[0022] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0023] Example 1 Screening of SNP Molecular Markers

[0024] By resequencing a total of 465 individuals from 15 Chinese local sheep breeds, the UNC5C gene was screened for Hu sheep and other breeds of sheep using selection signals and whole-genome association analysis methods, and the lamb number trait association analysis was performed on the Hu sheep with continuous single-multiple lamb breeding populations, and multiple SNP sites related to the sheep lamb number trait were discovered.

[0025] 1) Select signal localization gene

[0026] Different local sheep breeds were used, including the following samples:

[0027] Single lamb groups include: Tan sheep, Sunite sheep, Tibetan sheep PT, OL, red-headed sheep, Hulunbuir sheep, Bayinbuluke sheep, Sishui fur sheep, Altay sheep, and Bashbai.

[0028] The Hu sheep population includes: Hu sheep populations from 8 core breeding farms.

[0029] The selected segments related to lambing between Hu sheep and single lamb populations were located: Based on the LD 0.98 filtered data, the Fst statistic between Hu sheep and single lamb populations was calculated by sliding window, with a window size of 50K and a sliding window size of 25K each time. The top 1‰ was taken as the candidate window, and the significant sites obtained were as follows Figure 1 As shown, a candidate segment on chromosome 6 was located, which contains the UNC5C gene.

[0030] 2) Genome-wide association analysis to locate genes

[0031] Based on LD 0.98 filtering data, the Hu sheep phenotype was set to 1, and the single lamb group phenotype was set to 0. The tool saige was used to perform genome-wide association analysis on the binary phenotype. In the results, there was a significant signal on chr6, reaching the threshold. Site annotations with p values ​​less than 10e-6 were selected, and it was found that the UNC5C gene ( Figure 2 ).

[0032] 3) Continuous single-lamb and multiple-lamb data verification results

[0033] In order to better associate lambing phenotypes with genes, we selected 30 Hu sheep that gave birth to single lambs for more than three consecutive litters and 17 Hu sheep that gave birth to three lambs for more than three consecutive litters from nearly 800 ewes with continuous lambing records in Hu sheep breeding populations with similar genetic backgrounds for resequencing. Similarly, the top 1‰ sites of Fst and Pi-ratio between the continuous single lamb and continuous multiple lamb groups were used as candidates. Based on the continuous single and multiple lamb data, the Fst and Pi statistics between the single lamb group and the multiple lamb group were calculated with a sliding window, and the top 1‰ intersection of the two was taken as the candidate interval. The candidate interval includes the UNC5C variant site, such as Figure 3 shown.

[0034] By analyzing the annotation results of the signal-significant segment on Chr6, two SNP sites in the UNC5C gene in this segment were obtained. These two SNP sites were extremely significantly different in multi-lamb and single-lamb Hu sheep, and were significantly correlated with the lamb number trait of sheep.

[0035] The first SNP is located at position 29907419 of sheep chromosome 6 (located at position 149 of the nucleotide sequence shown in SEQ ID No.1), with a G / A polymorphism; the second SNP is located at position 29925629 of sheep chromosome 6 (located at position 188 of the nucleotide sequence shown in SEQID No.2), with a T / C polymorphism, and the reference genome version is NC_056059.1, ARS-UI_Ramb_v2.0.

[0036] The comparison of the number of lambs born and the number of lambs born alive of individuals with different genotypes at the same locus showed that for the Chr 6_29907419 locus, sheep with genotypes GG and AG had higher number of lambs born and the number of lambs born alive, among which the average number of lambs born and the number of lambs born alive of individuals with genotype GG was the highest ( Figure 4). For Chr 6_29925629, sheep with genotypes TT and TC had higher lambing numbers and lambs born alive, among which the average lambing number of individuals with genotype TT was the highest, and the average lambs born alive of individuals with genotype TC was the highest ( Figure 5 ).

[0037] Therefore, in sheep breeding, sheep with genotypes of Chr 6_29907419, GG and AG, and sheep with genotypes of Chr 6_29925629, TT and TC can be selected for breeding to improve reproductive efficiency and economic benefits.

[0038] Example 2 Detection of SNP site genotype

[0039] (1) For Chr 6_29907419 site, PCR amplification and sequencing were performed using the following primers:

[0040] U29907419-F:5'-ATAGGTGTTTTGTGCAGGCTG

[0041] U29907419-R:5'-AGAGAAGCATGTGTACACCTGA

[0042] We used high-fidelity PCR enzymes from Nanjing Novozyme Biotechnology Co., Ltd. Max MasterMix) for amplification, catalog number P525-02.

[0043] Chr 6_Chr 6_29907419 amplification system

[0044]

[0045] The amplification conditions are as follows: amplification using a PCR amplification instrument, first stage: 95℃ pre-denaturation for 5min; second stage: 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 45s, 34 cycles; third stage: 72℃ final extension for 5min, 4℃ storage. The amplified product length is 339bp. The PCR gel image results are as follows Figure 6 Shown: 1-6 are bands containing this site.

[0046] The product obtained by PCR amplification in the previous step was used for Sanger sequencing. The sequencing was performed by Shanghai Bioengineering Co., Ltd. After comparing the sequencing results with the reference gene sequence, the genotypes of different sites were recorded ( Figure 7 ).

[0047] (2) For the Chr 6_29925629 site, PCR amplification and sequencing were performed using the following primers:

[0048] U29925629-F:5'-TCAGACATGACTGAGCAACTAACA

[0049] U29925629-R:5'-AAGAACCAAGGGGTTCATCAGCA

[0050] We used high-fidelity PCR enzymes from Nanjing Novozyme Biotechnology Co., Ltd. Max MasterMix) for amplification, catalog number P525-02.

[0051] Chr 6_29925629 amplification system

[0052]

[0053]

[0054] The amplification conditions are as follows: amplification using a PCR amplification instrument, first stage: 95℃ pre-denaturation for 5min; second stage: 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 45s, 34 cycles; third stage: 72℃ final extension for 5min, 4℃ storage. The amplified product length is 339bp. The PCR gel image results are as follows Figure 8 Shown: 1-6 are bands containing this site.

[0055] The product obtained by PCR amplification in the previous step was used for Sanger sequencing. The sequencing was performed by Shanghai Bioengineering Co., Ltd. After comparing the sequencing results with the reference gene sequence, the genotypes of different sites were recorded ( Fig. 9 ).

Claims

1. A method for predicting the number of lambs born by sheep, characterized in that: The genotypes of the 29907419th site and / or the 29925629th site of chromosome 6 of the sample sheep were tested. The reference genome version was ARS-UI_Ramb_v2.

0. The genotypes of these two sites were used for prediction: When the genotype of the 29907419th site is GG or GA, the number of lambs born and the number of lambs born alive are higher, and the number of lambs born and the number of lambs born alive of the GG genotype are higher than those of the GA, and when the genotype is AA, the number of lambs born and the number of lambs born alive are lower; When the genotype of the 29925629th locus is TT or TC, the number of lambs born and the number of lambs born alive are higher, and the number of lambs born and the number of lambs born alive of the TT genotype are higher than those of the TC. When the genotype is CC, the number of lambs born and the number of lambs born alive are lower.

2. Application of a material for detecting the genotype of a SNP site in the prediction of lamb number traits in sheep or in the assisted breeding of lamb number traits in sheep, wherein there are two SNP sites, located on chromosome 6 of sheep, one of which is located at position 149 of the nucleotide sequence shown in SEQ ID No.1 and has a G / A polymorphism, and the other SNP is located at position 188 of the nucleotide sequence shown in SEQ ID No.2 and has a T / C polymorphism, When the genotype of the SNP site located at the 149th position of the nucleotide sequence shown in SEQ ID No. 1 is GG or GA, the number of lambs born and the number of lambs born alive are higher, and the number of lambs born and the number of lambs born alive of the GG genotype are higher than those of the GA genotype, and when the genotype is AA, the number of lambs born and the number of lambs born alive are lower; When the genotype of the SNP site located at the 188th position of the nucleotide sequence shown in SEQ ID No.2 is TT or TC, the number of lambs born and the number of lambs born alive are higher, and the number of lambs born and the number of lambs born alive of the TT genotype are higher than those of the TC. When the genotype is CC, the number of lambs born and the number of lambs born alive are lower.

3. The use according to claim 2, characterized in that: The substance is two pairs of primers, wherein the nucleotide sequence of one pair of primers is shown as SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequence of the other pair of primers is shown as SEQ ID No.5 and SEQ ID No.6.

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