A SNP locus genotype combination related to growth traits of Nubian goats and its application

Abstract

The present invention provides a SNP site genotype combination related to the growth traits of Nubian goats and its application, belonging to the field of animal molecular breeding technology. The SNP site genotype combination includes one or more of A1 to A6, and the genotypes corresponding to the SNP site genotype combination shown in A1 to A6 are B1 to B6 respectively; the genotype of the B1 is AGGA type, the genotype of the B2 is one of AGTT type and AATT type, the genotype of the B3 is AGCG type, the genotype of the B4 is one of GGTC type, GATT type and GATC type, the genotype of the B5 is one of AACC type, GACG type and GGCG type, and the genotype of the B6 is one of TCCC type, TTCG type and TTGG type. By screening using the SNP site genotype combination of the present invention, Nubian goat individuals with excellent growth traits can be quickly identified.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal molecular breeding, and in particular relates to a SNP site genotype combination related to the growth traits of Nubian goats and an application thereof. Background Art

[0002] The importance of specific morphological measurements in goats is paramount, as they serve as crucial indicators of skeletal development, overall health, and reproductive success. Parameters such as body height, body length, body length, and canal circumference are key to assessing skeletal development, providing valuable information on bone growth and structural integrity. Furthermore, careful assessment of chest circumference, abdominal circumference, and hindquarter circumference not only provides insights into skeletal development but is also essential for studying goat fattening. These measurements provide a comprehensive understanding of skeletal development and highlight the intricate interrelationships between various anatomical features. Rump width is a multifaceted parameter, playing a dual role in skeletal development and fattening in goats. Beyond its influence on skeletal size, rump width is also crucial for the reproductive success of ewes. Rump width has been shown to significantly influence the lambing process, with its measurement closely correlated with overall farrowing success. When evaluating reproductive performance in ewes, lamb number is the most important indicator. This critical benchmark not only summarizes a ewe's reproductive efficiency but also reflects the success of a breeding program. Therefore, the body measurement data of body height, oblique body length, straight body length, chest circumference, abdominal circumference, tube circumference and rump width, as well as the number of lambs, were jointly analyzed with the candidate gene SNPs, and these morphological parameters were comprehensively evaluated, which not only helps to understand the growth and development of goats, but also plays a vital role in measuring the reproductive potential of goat populations.

[0003] The NDRG4 gene is a member of the N-myc downregulated gene family. Current studies indicate that the NDRG family plays important roles in cell biology, including inhibiting cell proliferation, inhibiting tumor cell migration, and promoting cell differentiation. Functional studies of NDRG4 have revealed that this gene may be involved in regulating cell differentiation, proliferation, and apoptosis. Studies have shown that NDRG4 deficiency can cause G1 cell cycle arrest and apoptosis. Studies of NDRG4 gene polymorphisms in porcine pigs have shown a significant association between the rs322396417 polymorphism and the number of teats per nipple in French Large White pigs, and a highly significant association between the rs325739056 polymorphism and backfat thickness in French Large White pigs. In regulating muscle development, NDRG4 binding to carboxyl-terminal regulatory protein (CTMP) weakens the interaction between CTMP and protein kinase B (Akt), increasing the phosphorylation of Akt and cAMP response element binding protein (CREB), leading to increased expression of myogenic genes. It was demonstrated that NDRG4 promoted myogenic differentiation through Akt / CREB activation.

[0004] Although there are many studies on the association analysis of body size data or lambing data with candidate gene SNPs in goats, there are few studies on the association analysis of NDRG4 gene SNPs with body size data and lambing data at the same time. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a SNP site genotype combination related to the growth traits of Nubian goats and its application, wherein the SNP site genotype combination can be used to breed Nubian goats with excellent body size traits.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a SNP site genotype combination associated with the growth traits of Nubian goats, wherein the SNP site genotype combination includes one or more of A1 to A6, and the genotypes corresponding to the SNP site genotype combination represented by A1 to A6 are B1 to B6 respectively;

[0008] A1 is g. 27632588 A>G and g. 27640465 G>A;

[0009] A2 is g. 27632588 A>G and g. 27640826 T>C;

[0010] A3 is g. 27632588 A>G and g. 27642312 C>G;

[0011] A4 is g. 27640465 G>A and g. 27640826 T>C;

[0012] A5 is g. 27640465 G>A and g. 27642312 C>G;

[0013] A6 is g. 27640826 T>C and g. 27642312 C>G;

[0014] The g. 27632588 A>G indicates that the base 27632588 on chromosome 18 of the NDRG4 gene is A / G. The GenBank accession number of the NDRG4 gene is 102176452.

[0015] The g. 27640465 G>A means that the base 27640465 on chromosome 18 of the NDRG4 gene is G / A. The GenBank accession number of the NDRG4 gene is 102176452.

[0016] The g. 27640826 T>C means that the base 27640826 on chromosome 18 of the NDRG4 gene is T / C. The GenBank accession number of the NDRG4 gene is 102176452;

[0017] The g. 27642312 C>G indicates that the base 27642312 on chromosome 18 of the NDRG4 gene is C / G. The GenBank accession number of the NDRG4 gene is 102176452.

[0018] The genotype of the B1 is AGGA, the genotype of the B2 is one of AGTT and AATT, the genotype of the B3 is AGCG, the genotype of the B4 is one of GGTC, GATT and GATC, the genotype of the B5 is one of AACC, GACG and GGCG, and the genotype of the B6 is one of TCCC, TTCG and TTGG.

[0019] The present invention also provides an application of the above-mentioned SNP site genotype combination in improving the growth traits of Nubian goats.

[0020] The present invention also provides a method for improving the growth traits of Nubian goats, comprising the following steps: detecting the nucleotides of each SNP site in the above-mentioned SNP site genotype combination in the genome of the sheep to be tested, determining the genotype of the sample to be tested corresponding to the SNP site genotype combination, and if the genotype of the SNP site genotype combination of the sheep to be tested belongs to at least one of the above-mentioned B1 to B6, the sheep to be tested is a Nubian goat with high growth traits.

[0021] The present invention also provides an application of a substance for detecting nucleotides at each SNP site in the above-mentioned SNP site genotype combination in screening Nubian goats with high growth traits.

[0022] Preferably, the substance is a PCR primer and / or sequencing primer designed for the SNP site to be detected.

[0023] Preferably, the primers for detecting g. 27632588 A>G are shown in SEQ ID NO.1 to SEQ ID NO.2;

[0024] The primers used to detect g. 27640465 G>A are shown in SEQ ID NO. 9 to SEQ ID NO. 10;

[0025] The primers used to detect g. 27640826 T>C are shown in SEQ ID NO. 11 to SEQ ID NO. 12;

[0026] The primers used to detect g. 27642312 C>G are shown in SEQ ID NO. 13 to SEQ ID NO. 14.

[0027] The present invention also provides a primer combination, which includes a first primer combination to a sixth primer combination;

[0028] The first primer combination to the sixth primer combination include primers for detecting the nucleotides of each SNP site in A1 to A6 above;

[0029] The primers used to detect g. 27632588 A>G are shown in SEQ ID NO. 1 to SEQ ID NO. 2;

[0030] The primers used to detect g. 27640465 G>A are shown in SEQ ID NO. 9 to SEQ ID NO. 10;

[0031] The primers used to detect g. 27640826 T>C are shown in SEQ ID NO. 11 to SEQ ID NO. 12;

[0032] The primers used to detect g. 27642312 C>G are shown in SEQ ID NO. 13 to SEQ ID NO. 14.

[0033] The present invention also provides an application of the primer combination in Nubian goat breeding.

[0034] The present invention also provides an application of the primer combination in screening SNP site genotype combinations related to growth traits of Nubian goats.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a single-nucleotide polymorphism (SNP) genotype combination associated with growth traits of Nubian goats and its application. The SNP genotype combination provided by the present invention is significantly correlated with the growth traits of Nubian goats. Primers designed for this SNP genotype combination can be used in Nubian goat breeding to screen for goats with superior growth traits. Using this SNP genotype combination for screening can rapidly identify individual Nubian goats with excellent growth traits, thereby improving the economic value and market competitiveness of Nubian goats. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The results of goat genomic DNA gel electrophoresis are shown, where M is a 5000 bp marker and 1 to 10 are 10 randomly selected goat DNA samples.

[0038] Figure 2 This is the gel electrophoresis result of the amplification products of NDRG4 gene with four pairs of primers: N4, N17-1, N17-2 and N17-3;

[0039] Figure 3 The electrophoresis detection results of NDRG4 gene digested with HhaI, where M is a 1000 bp marker;

[0040] Figure 4 This is the high-resolution melting curve of the g. 27640465 G>A site of the NDRG4 gene;

[0041] Figure 5 This is the high-resolution melting curve of the g. 27640826 T>C site of the NDRG4 gene;

[0042] Figure 6 This is the high-resolution melting curve of the g. 27642312 C>G site of the NDRG4 gene;

[0043] Figure 7 The results of direct sequencing of SNPs for the NDRG4 gene;

[0044] Figure 8 Linkage disequilibrium analysis of 4 SNPs in Leizhou goats, Sichuan black goats and Nubian goats. A is the linkage disequilibrium analysis of 4 SNPs in Leizhou goats (LZ), B is the linkage disequilibrium analysis of 4 SNPs in Sichuan black goats (CZ), and C is the linkage disequilibrium analysis of 4 SNPs in Nubian goats (NS). The numbers in the figure represent r 2 Value (%), 32588 A>G represents g. 27632588 A>G, 40465 G>A represents g. 27640465 G>A, 40826 T>C represents g. 27640826 T>C, 42312 C>G represents g. 27642312 C>G. DETAILED DESCRIPTION

[0045] The present invention provides a SNP site genotype combination associated with the growth traits of Nubian goats, wherein the SNP site genotype combination includes one or more of A1 to A6, and the genotypes corresponding to the SNP site genotype combination represented by A1 to A6 are B1 to B6 respectively;

[0046] A1 is g. 27632588 A>G and g. 27640465 G>A;

[0047] A2 is g. 27632588 A>G and g. 27640826 T>C;

[0048] A3 is g. 27632588 A>G and g. 27642312 C>G;

[0049] A4 is g. 27640465 G>A and g. 27640826 T>C;

[0050] A5 is g. 27640465 G>A and g. 27642312 C>G;

[0051] A6 is g. 27640826 T>C and g. 27642312 C>G;

[0052] The g. 27632588 A>G indicates that the base 27632588 on chromosome 18 of the NDRG4 gene is A / G. The GenBank accession number of the NDRG4 gene is 102176452.

[0053] The g. 27640465 G>A means that the base 27640465 on chromosome 18 of the NDRG4 gene is G / A. The GenBank accession number of the NDRG4 gene is 102176452.

[0054] The g. 27640826 T>C means that the base 27640826 on chromosome 18 of the NDRG4 gene is T / C. The GenBank accession number of the NDRG4 gene is 102176452;

[0055] The g. 27642312 C>G indicates that the base 27642312 on chromosome 18 of the NDRG4 gene is C / G. The GenBank accession number of the NDRG4 gene is 102176452.

[0056] The genotype of the B1 is AGGA, the genotype of the B2 is one of AGTT and AATT, the genotype of the B3 is AGCG, the genotype of the B4 is one of GGTC, GATT and GATC, the genotype of the B5 is one of AACC, GACG and GGCG, and the genotype of the B6 is one of TCCC, TTCG and TTGG.

[0057] The results of this study showed that in Nubian goats, the AGGA genotype at the g. 27632588 A>G and g. 27640465 G>A loci combined had significantly higher chest and abdominal circumferences than the AAAA genotype (P<0.01). In single-locus analysis, the AG genotype also had significantly higher chest and abdominal circumferences than the AA genotype (P<0.05, P<0.01). At the g. 27640465 G>A locus, the average chest circumference of the GA genotype was significantly higher than that of the AA genotype (P<0.05). This suggests an additive effect at these two loci, meaning that the genotype combination at these two loci has an additive effect on body size traits such as chest circumference and abdominal circumference, resulting in a more significant impact.

[0058] Combination analysis of the g. 27632588 A>G and g. 27640826 T>C loci in Nubian goats revealed that the AGTT genotype had significantly higher chest and abdominal circumferences than the AATC and AATT genotypes (P < 0.05). Furthermore, the AGTT genotype had a significantly higher rump width than the AATC genotype (P < 0.01) and significantly higher than the AATT, AGCC, AGTC, and GGTT genotypes (P < 0.05). Furthermore, the AATT genotype had a significantly higher hindquarter circumference than the AGTC genotype (P < 0.05). Among reproductive traits, the GGTT genotype had the lowest litter size in parity 1, which was significantly lower than the AATT genotype (P < 0.01) and significantly lower than the AGCC, AGTC, and AGTT genotypes (P < 0.05). This indicates that the genotype combination at these two loci significantly affects both body size and reproductive traits. In single-locus analysis, the chest and abdominal circumferences of Nubian goats with the g. 27632588 A>G locus were significantly greater in AG-type Nubian goats than in AA-type individuals (P < 0.05, P < 0.01). Furthermore, the number of lambs per litter was significantly lower in GG-type Nubian goats than in AA and AG-type Nubian goats (P < 0.05). In the g. 27640826 T>C locus analysis, the average level of hind leg circumference in TT-type Nubian goats was significantly greater than that in TC-type Nubian goats (P < 0.05). Furthermore, the average level of rump width in TT-type Nubian goats was significantly greater than that in CC-type Nubian goats (P < 0.05). This suggests that the genotype at a single locus also has a certain influence on body size and reproductive traits. Thus, there is an additive effect between the g. 27632588 A>G and g. 27640826 T>C loci on both body size and reproductive traits. Among body size and reproductive traits, the AGTT genotype performed best in chest circumference, abdominal circumference, and rump width, while the GGTT genotype performed worst in reproductive traits. Therefore, the effects of these two loci on these traits may be the most significant, and the AGTT genotype may be the dominant combination genotype.

[0059] Combined genetic analysis of the g. 27632588 A>G and g. 27642312 C>G loci in Nubian goats revealed that, for chest circumference, the AGCG genotype was significantly higher than the AACG and AAGG genotypes (P < 0.01), and significantly higher than the AACC, AGGG, and GGGG genotypes (P < 0.05). For abdominal circumference, the AGCG genotype was also significantly higher than the AAGG genotype (P < 0.01), while the AGCC and AGGG genotypes were also significantly higher than the AAGG genotype (P < 0.05). In single-locus analysis, the chest and abdominal circumference of AG-type Nubian goats at the g. 27632588 A>G locus were significantly higher than those of AA-type individuals (P < 0.05, P < 0.01). At the g. 27642312 C>G locus, CC-type individuals had significantly larger chest circumference than both CG-type and GG-type individuals (P < 0.05). This indicates that the genotypes at these two loci have an additive effect on the body size traits of chest circumference and abdominal circumference, with the combined effect being greater than that of a single locus, consistent with an additive effect. Furthermore, the significant dominance of the AGCG genotype in chest circumference and abdominal circumference suggests that this combined genotype may have the greatest impact on these two traits, while the significant dominance of the CC genotype in chest circumference suggests that this genotype may be the dominant genotype at this locus.

[0060] Combined genetic analysis of the g. 27640465 G>A and g. 27640826 T>C loci in Nubian goats revealed that the GGTC genotype exhibited significantly higher body height than the GGTT and AATC genotypes (P < 0.05). In terms of chest circumference, the GATC genotype exhibited significantly higher chest circumference than the AATC genotype (P < 0.05). In terms of hindquarter circumference, the GATT genotype exhibited significantly higher hindquarter circumference than the GATC genotype (P < 0.01) and significantly higher hindquarter circumference than the AATC, GGTC, and GGTT genotypes (P < 0.05). In terms of canthus circumference, the GATT genotype exhibited significantly higher canthus circumference than the AATC and AATT genotypes (P < 0.05). In single-locus analysis, at the g. 27640465 G>A locus, the average level of body slant length in GA genotype individuals was significantly higher than that in GG genotype individuals (P < 0.05). Furthermore, the average levels of chest circumference and canthus circumference in GA genotype individuals were significantly higher than those in AA genotype individuals (P < 0.05). The average level of rump width of GG type individuals was significantly higher than that of AA type individuals (p < 0.05).

[0061] At the g. 27640826 T>C locus, CC-type individuals showed significantly greater height than TC-type and TT-type individuals (p < 0.01). Furthermore, TT-type individuals had significantly greater hind leg circumference than TC-type individuals (p < 0.05). Furthermore, the average level of rump width in TT-type individuals was significantly greater than that in CC-type individuals (p < 0.05). These results suggest that these two loci may have an additive effect on body size traits in goats.

[0062] Combined genetic analysis of the g. 27640465 G>A and g. 27642312 C>G loci in Nubian goats revealed that for body size traits, the AACC and GACG genotypes were significantly higher than the AAGG genotype (P<0.01). Furthermore, the AACC genotype was significantly higher than the AACG and GGCG genotypes (P<0.01). For canine circumference, the GACG genotype was significantly higher than the AACG genotype (P<0.01) and significantly higher than the GACC and GGCG genotypes (P<0.05). For rump width, the GGCG and GGGG genotypes were significantly higher than the AACG genotype (P<0.05). Single-locus analysis revealed that at the g. 27640465 G>A locus, the average levels of chest and canine circumference in GA genotypes were significantly higher than those in AA genotypes (P<0.05). The average level of rump width in GG genotypes was significantly higher than that in AA genotypes (P<0.05). At the g. 27642312C>G locus, CC individuals had significantly larger chest circumference than both CG and GG individuals (p < 0.05). These results suggest an additive effect between these two loci, with the most significant effect on chest circumference. Furthermore, AACC individuals exhibited an advantage in chest circumference, potentially representing a dominant genotype.

[0063] Combined genetic analysis of the g. 27640826 T>C and g. 27642312 C>G loci in Nubian goats revealed that for chest circumference, the TCCC and TTCC types were significantly higher than the TTCG type (P < 0.05). For hind leg circumference, the TTCG and TTGG types were both extremely significantly higher than the TCCG type (P < 0.01) and significantly higher than the TTCC type (P < 0.05). Analysis of individual loci revealed that at the g. 27640826 T>C locus, the TT type had significantly higher hind leg circumference than the TC type (P < 0.05). At the g. 27642312 C>G locus, the CC type had significantly larger chest circumference than both the CG and GG types (P < 0.05). These results suggest an additive effect between these two loci.

[0064] The present invention also provides an application of the above-mentioned SNP site genotype combination in improving the growth traits of Nubian goats.

[0065] The present invention also provides a method for improving the growth traits of Nubian goats, comprising the following steps: detecting the nucleotides of each SNP site in the above-mentioned SNP site genotype combination in the genome of the sheep to be tested, determining the genotype of the sample to be tested corresponding to the SNP site genotype combination, and if the genotype of the SNP site genotype combination of the sheep to be tested belongs to at least one of the above-mentioned B1 to B6, the sheep to be tested is a Nubian goat with high growth traits.

[0066] The present invention also provides an application of a substance for detecting nucleotides at each SNP site in the above-mentioned SNP site genotype combination in screening Nubian goats with high growth traits.

[0067] In the present invention, the growth traits include body size traits, and the body size traits include one or more of chest circumference, abdominal circumference, buttocks width, hind leg circumference, and tube circumference.

[0068] In the present invention, the substances are PCR primers and / or sequencing primers designed for the SNP sites to be detected. In a preferred embodiment, primers for detecting g. 27632588 A>G are shown in SEQ ID NOs. 1 to 2; primers for detecting g. 27640465 G>A are shown in SEQ ID NOs. 9 to 10; primers for detecting g. 27640826 T>C are shown in SEQ ID NOs. 11 to 12; and primers for detecting g. 27642312 C>G are shown in SEQ ID NOs. 13 to 14.

[0069] The present invention also provides a primer combination, which includes a first primer combination to a sixth primer combination;

[0070] The first primer combination to the sixth primer combination include primers for detecting the nucleotides of each SNP site in A1 to A6 above;

[0071] The primers used to detect g. 27632588 A>G are shown in SEQ ID NO. 1 to SEQ ID NO. 2;

[0072] The primers used to detect g. 27640465 G>A are shown in SEQ ID NO. 9 to SEQ ID NO. 10;

[0073] The primers used to detect g. 27640826 T>C are shown in SEQ ID NO. 11 to SEQ ID NO. 12;

[0074] The primers used to detect g. 27642312 C>G are shown in SEQ ID NO. 13 to SEQ ID NO. 14.

[0075] In the present invention, the first primer combination is a primer for detecting nucleotides g. 27632588 A>G and g. 27640465 G>A; the second primer combination is a primer for detecting nucleotides g. 27632588 A>G and g. 27640826 T>C; the third primer combination is a primer for detecting nucleotides g. 27632588 A>G and g. 27642312 C>G; the fourth primer combination is a primer for detecting nucleotides g. 27640465 G>A and g. 27640826 T>C; the fifth primer combination is a primer for detecting nucleotides g. 27640465 G>A and g. 27642312 C>G; and the sixth primer combination is a primer for detecting nucleotides g. 27640826 T>C and g. 27642312 primers with nucleotides C>G.

[0076] The present invention also provides an application of the primer combination in Nubian goat breeding.

[0077] The present invention also provides an application of the primer combination in screening SNP site genotype combinations related to growth traits of Nubian goats.

[0078] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0079] Example 1

[0080] 1. Experimental Materials

[0081] 1.1 Experimental samples

[0082] The present invention uses three breeds of goats, namely Leizhou goats, Sichuan black goats and Nubian goats, as experimental materials, selects healthy adult ewes for body measurement, lambing data collection and blood sample collection.

[0083] Goat body measurements were manually performed using tools such as a measuring tape, a circular measuring device, and a measuring rod. Following each goat's ear tag number, measurements were taken of body height, body length, body oblique length, chest circumference, abdominal circumference, hind leg circumference, leg circumference, and rump width. For measurement, the goats were required to stand upright on a flat surface. One person held the goat in place while another took and recorded measurements. A total of 721 complete and valid goat body measurement data sets were used in this experiment, including 224 from Leizhou goats, 407 from Sichuan Black goats, and 90 from Nubian goats.

[0084] In addition, a total of 951 complete and valid goat lambing data were used in this experiment, including 305 Leizhou goats, 523 Sichuan black goats, and 123 Nubian goats.

[0085] Use disposable vacuum negative pressure blood collection tubes (anticoagulant tubes) to collect blood from the goat's jugular vein (usually from the left jugular vein). Collect approximately 5 mL of whole blood from each goat. After blood collection, shake the blood with the anticoagulant to achieve anticoagulation. Place the blood collection tube in a blood collection rack (place the rack in a foam box with plenty of ice to ensure the blood sample is kept refrigerated). Once blood collection is complete, bring the blood back to the laboratory and freeze it in a -80°C ultra-low temperature freezer.

[0086] 1.2 Various enzymes, reagents and kits

[0087] Table 1 Enzymes, reagents and kits used in this experiment

[0088]

[0089] 1.3 Experimental Methods

[0090] 1.3.1 Extraction and quality testing of blood genomic DNA

[0091] This study used the Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) for DNA extraction. After DNA extraction according to the kit's instructions, the extracted DNA fragments were assayed for concentration and purity using a NanoDrop spectrophotometer. A blank control of 2 μL of ddH₂O was used to ensure that the extracted DNA sample concentration was no less than 30 ng / μL and that the OD₂₀ / OD₂₀ values ​​were between 1.8 and 2.0. The quality of the extracted DNA was then assessed by electrophoresis on a 1% agarose gel.

[0092] 1.3.2 Primer design, PCR amplification, and product sequencing

[0093] (1) Construction of DNA pool

[0094] After the extracted genomic DNA was analyzed for concentration and purity using a UV spectrophotometer, 9, 9, 18, 18, and 27 DNA samples were collected from each variety. 11.5, 11.5, 6.0, 6.0, and 4.0 μL of each sample were taken, respectively, to construct 15 DNA pools. Each pool consisted of approximately 100 μL. These were stored at -20°C and used for screening of candidate gene SNPs.

[0095] (2) Design of candidate gene primers

[0096] Based on the candidate gene sequence published in GenBank (http: / / www.ncbi.nlm.nih.gov / ) (accession number 102176452), the following primers were designed using Primer Premier 5.0 software. The primers were synthesized by Beijing Liuhe BGI Genomics Co., Ltd. The primer information is shown in Table 2.

[0097] Table 2 Primer information used for SNPs detection

[0098]

[0099] (3) PCR amplification procedure

[0100] Table 3 PCR reaction system and procedure

[0101]

[0102] According to the PCR reaction system and procedure in Table 3, the DNA sample was amplified by PCR amplification instrument. After the gel electrophoresis test was qualified, the obtained amplified product was collected and retained for the next enzyme digestion reaction or HRM reaction.

[0103] (4) PCR product sequencing and sequence alignment

[0104] After gel electrophoresis, PCR products with high specificity and yield were sequenced by Beijing Liuhe BGI Genomics Co., Ltd., using bidirectional sequencing for each product. After sequencing, upstream and downstream sequences were obtained, and sequence splicing was performed using DNAStar software. SNPs were then screened based on the sequencing peaks.

[0105] 2.3.3 RFLP detection

[0106] Use Primer Premier 5.0 software to search for restriction enzymes corresponding to the screened SNP sites. If there are no restriction sites or the restriction enzymes are too expensive, use a creative enzyme digestion method to introduce mismatched bases into the primers. Then, use the corresponding restriction enzymes to digest the PCR amplification products of the candidate gene polymorphic sites. Configure the enzyme digestion reaction system according to Table 4. After thorough vortexing and mixing, place the reaction in a 37°C constant temperature water bath for enzyme digestion for 30 minutes. After digestion is complete, remove the sample and perform gel electrophoresis. Analyze the genotype presented in each lane based on the gel imaging results.

[0107] Table 4 Enzyme digestion reaction system

[0108]

[0109] 1.3.4 High-resolution melting curve (HRM)

[0110] 1.3.4.1 Design and synthesis of high-resolution melting curve PCR primers

[0111] Primers were designed using Primer Premier 5.0 software based on candidate gene sequences published in GenBank (http: / / www.ncbi.nlm.nih.gov / ), and synthesis was performed by Beijing Liuhe BGI Genomics Co., Ltd. Detailed information on primer sequences is provided in Table 2.

[0112] 1.3.4.2 High-resolution melting curve reaction conditions

[0113] A temperature gradient of 55-65°C was used to determine the optimal annealing temperature of the primers and obtain specific PCR products, thereby increasing the sensitivity of high-resolution melting.

[0114] Table 5 PCR reaction system and procedure

[0115]

[0116] The F / R in Table 5 are primers N17-1 F (SNP2) / N17-1 R (SNP2), N17-2 F (SNP3) / N17-2 R (SNP3), and N17-3 F (SNP4) / N17-3 R (SNP4), respectively.

[0117] The HRM reaction program was as follows: 95°C for 15 s, 55°C for 15 s, and then 55°C to 95°C (temperature ramp rate of 0.1°C / s). Fluorescence changes were continuously monitored, and data were collected to generate melting curves. The results were analyzed using LightCycler 480 Software release 1.5.1.62.

[0118] 1.4 Major Molecular Biology Software

[0119] PCR primer design software: Primer Premier 5.0 was used for primer design;

[0120] Homology sequence analysis software: DNAStar is used for DNA sequence splicing and translation, sequencing peak analysis, etc.

[0121] Restriction enzyme cutting site analysis: SnapGene is used for restriction enzyme cutting analysis;

[0122] Linkage disequilibrium and allele frequency analysis: http: / / analysis.bio-x.cn / myAnalysis.php;

[0123] Nucleic acid sequence database: http: / / www.ncbi.nlm.nih.gov (GenBank);

[0124] BLAST program: http: / / www.ncbi.nlm.nih.gov / BLAST / was used for sequence alignment;

[0125] 1.5 Statistical methods in population genetics

[0126] 1.5.1 Gene frequency and genotype frequency

[0127] Gene frequency refers to the ratio of a gene to all genes at the same locus in a population.

[0128] Gene frequencies are represented by p and q respectively, and the calculation formula is:

[0129] P=(2nBB +nAB) / (2(nBB + nAB + nAA))

[0130] q=(2nAA + nAB) / (2(nBB + nAB + nAA))

[0131] Where: p is the gene frequency of allele B, q is the gene frequency of allele A. nBB, nAB and nAA are the number of individuals with genotype BB, AB and AA in the population respectively.

[0132] Genotype frequency refers to the ratio of individuals of a particular genotype to the total number of individuals in a diploid population. If the PCR-RFLP test result indicates a codominant allele, the genotype frequency can be calculated as follows:

[0133] Genotype frequency = number of genotypes / total number of tests.

[0134] 1.5.2 Testing for Hardy-Weinberg Equilibrium in a Population

[0135] Hardy-Weinberg equilibrium refers to the state in which genotype frequencies remain stable throughout the alternation of generations in an infinite or sufficiently large, closed, randomly mating Mendelian population. This stable state is called Hardy-Weinberg equilibrium. For a population in Hardy-Weinberg equilibrium, when a locus has two alleles, the following formula holds:

[0136] P=p² H=2pq Q=q²

[0137] nAA′ = (nBB + nAB + nAA)P

[0138] nAB′ = (nBB + nAB + nAA)H

[0139] nBB′ = (nBB + nAB + nAA)Q

[0140] Wherein nBB′, nAB′, and nAA′ in the formula are the theoretical values ​​of nBB, nAB, and nAA.

[0141] The χ² test of fitness is used to determine whether a population is in equilibrium (Li Chunxi, Wang Zhihe, Wang Wenlin. Biostatistics - 2nd Edition [M]. Science Press, 2000). The calculation formula is as follows:

[0142]

[0143] When df=1 and the sample size is small, especially when the theoretical number of times is less than 5, continuity correction is required. The corrected formula is as follows:

[0144]

[0145] Where: A is the actual observed value, T is the theoretical observed value, n is the number of alleles, and df is the degree of freedom. 1.5.3 Genetic homozygosity

[0146] Genetic homozygosity (closed-tube method for differentiating homozygotes and heterozygotes [J]. Clinical Chemistry, 2003, 49(3):396-406.) refers to the degree of homozygosity of alleles at a particular locus in a population. The formula for calculating homozygosity at a locus in a population is:

[0147]

[0148] In the formula, Pᵢ is the frequency of the i-th allele at a certain gene locus, n is the number of alleles at a certain locus, and Ho is the homozygosity of a certain locus.

[0149] 1.5.4 Genetic heterozygosity

[0150] The concept opposite to genetic homozygosity is genetic heterozygosity (closed-tube method for differentiating homozygotes and heterozygotes [J]. Clinical Chemistry, 2003, 49(3): 396-406.), which is the proportion of heterozygotes at a certain gene locus in a population. The genetic variation of a population can usually be measured by the proportion of polymorphic loci and the average heterozygosity of each locus. Therefore, average heterozygosity is an effective indicator for measuring genetic variation within a population. The greater the average heterozygosity, the greater the degree of genetic variation within the population. The formula for calculating the heterozygosity of a certain locus in a population is:

[0151]

[0152] In the formula, Pᵢ is the frequency of the i-th allele at a certain gene locus, n is the number of alleles at a certain locus, and He is the heterozygosity at a certain locus.

[0153] 1.5.5 Effective number of alleles

[0154] The effective allele number (Bansal A, Boom DVD, Kammerer S, et al. Association Testing by DNA Pooling: An Effective Initial Screen [J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(26):16871.) is the reciprocal of genetic homozygosity, reflecting the interaction between alleles and serving as another indicator of gene homozygosity. The more evenly alleles are distributed in a population, the closer the effective allele number is to the actual observed value. The formula for calculating the effective allele number at a certain locus in a population is:

[0155]

[0156] Where Pᵢ represents the frequency of the i-th allele, and n is the number of alleles.

[0157] 1.5.6 Polymorphic Information Content is Worth Calculating

[0158] The polymorphic information content (PIC) is used to estimate the polymorphism of marker genes. According to the classification criteria proposed by Botstein (Botstein D, White R L, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. American Journal of Human Genetics, 1980, 32(3):314-331.), PIC > 0.5 indicates high polymorphism, 0.25 < PCI < 0.5 indicates moderate polymorphism, and PIC < 0.25 indicates low polymorphism. The PIC value of a marker gene in a population is calculated based on the frequencies of its alleles and can be calculated using the following formula:

[0159]

[0160] where n is the number of alleles, and Pi and Pj are the frequencies of the i-th and j-th alleles in the population, respectively.

[0161] 1.6 Linkage Disequilibrium

[0162] Linkage disequilibrium refers to the situation in a population where the probability of alleles at two or more loci appearing on the same chromosome simultaneously is higher than the random occurrence frequency. Linkage disequilibrium is the frequency of alleles or genetic markers in a population that shows a higher or lower frequency than the expected frequency of a single modal sample based on the random frequencies of alleles.

[0163]

[0164] D’ (standardized disequilibrium coefficients) was proposed by Lewontin to obtain D’ by standardizing the D value. D’ reflects the recombination history of the population and is suitable for studying the degree of linkage disequilibrium in the population. The value range of D’ is from -1 to 1.

[0165]

[0166] " Among them:

[0167]

[0168] Using the correlation coefficient r 2(squared allele-frequency correlations) to measure the degree of linkage disequilibrium:

[0169]

[0170] r 2 Reflects the degree of allele correlation, with a value ranging from 0 to 1.

[0171] When D'=0, r 2 =0, it is in a state of complete linkage equilibrium; when D'=1, r 2 = 1, it is in a completely unlinked equilibrium state; if r 2 >0.33, it can be considered that there is strong linkage disequilibrium between the sites.

[0172] 1.7 Data Statistics and Analysis

[0173] 1.7.1 Data Analysis of Gene Polymorphisms

[0174] Statistical analysis of all loci in goats was performed using IBM SPSS Statistics 27 (General Linear Model, GLM) software with a constructed single-site effect model. Multiple comparisons of means were performed using the LSD and Dunnett's T3 methods. Results are presented as mean ± standard deviation. The following linear model was used for statistical analysis:

[0175] Analysis of single SNP loci on lamb size in sheep:

[0176] (Model 1)

[0177] Where Y ijk is the observed value of the trait; μ is the overall mean; G i is the genotype effect of marker i; ε ijk is the random residual effect.

[0178] 1.7.2 Statistical Analysis of Fluorescence Quantification Data

[0179] IBM SPSS Statistics 27 was used to calculate the Ct values ​​and standard deviations between repeated samples. The 2-ΔΔCt method was used to process the data and analyze the gene expression. ΔCt = Ct (candidate gene) - Ct (β-actin), 2 -ΔΔCt The expression of candidate genes is expressed as fold change relative to β-actin.

[0180] 2 Results and Analysis

[0181] 2.1 Detection of goat genomic DNA

[0182] Ten goat genomic DNA samples were randomly selected and tested by NanoDrop nucleic acid analyzer. The OD260 / OD280 values ​​of the goat genomic DNA samples were found to be between 1.8 and 2.0, and the concentrations were between 50 and 200 ng / µL. The samples were then tested by 1% agarose gel electrophoresis (e.g. Figure 1 ), the electrophoresis bands were clear and dense bright bands, indicating that the extracted goat genomic DNA sample was good, the purity and concentration met the test requirements, and could be used for subsequent experiments.

[0183] 2.2 Comparison of body size and lambing traits among goat breeds

[0184] Tables 6 and 7 show a comparative analysis of body size and litter size traits among Leizhou, Sichuan Black, and Nubian goats. Sichuan Black goats exhibited significantly greater body size traits than Leizhou and Nubian goats (P < 0.01). Nubian goats exhibited significantly greater body size traits, except for abdominal circumference, than Leizhou goats (P < 0.01). Furthermore, the litter size of Sichuan Black goats in their first, second, and third parities was significantly greater than that of Leizhou and Nubian goats (P < 0.01). Furthermore, the litter size of Leizhou goats in their first, second, and third parities was significantly greater than that of Nubian goats (P < 0.01). This indicates significant differences in body size and litter size among the three breeds.

[0185] Table 6 Comparative analysis of body size traits among different breeds of goats

[0186]

[0187] Note: Data in the same column marked with different capital letters differed significantly (P < 0.01), data marked with different lowercase letters differed significantly (P < 0.05), and data with no shoulder letters or with the same shoulder letters did not differ significantly (P > 0.05). LZ stands for Leizhou goat, CZ for Sichuan black goat, and NS for Nubian goat. This notation also applies to the following tables.

[0188] Table 7 Comparative analysis of lambing traits among different breeds of goats

[0189]

[0190] 2.3 NDRG4 gene detection results

[0191] Four pairs of primers (N4, N17-1, N17-2, N17-3, N17-4) of the NDRG4 gene were amplified, and the results are as follows Figure 2 As shown in the figure, the length of the amplified fragment is consistent with the target fragment and the band is clear without any other bands, so it can be directly used in the next RFLP experiment.

[0192] 2.3.1 PCR-RFLP analysis of the NDRG4 gene

[0193] DNA from three goat breeds was pooled and PCR amplified for the NDRG4 gene. After gel electrophoresis, the amplified products, which showed high specificity and yield, were sequenced by Beijing Liuhe BGI Genomics Co., Ltd., using bidirectional sequencing for each product. After sequencing, upstream and downstream sequences were obtained, and sequence assembly was performed using DNAStar software. SNPs were then screened based on the sequencing peaks.

[0194] Sequencing results showed that the NDRG4 gene (GenBank accession number 102176452) has a single base mutation in intron 3. The mutation occurred at bp 27632588 of the NDRG4 gene, with an A→G mutation, and was therefore named the g.27632588 A>G mutation.

[0195] SnapGene software was used to determine that the restriction endonuclease corresponding to the mutation site g. 27632588 A>G was HhaI. PCR amplification was performed with primer N4 (SEQ ID NOs. 1-2). The amplified product was digested with HhaI restriction endonuclease and analyzed by 2% agarose gel electrophoresis. The results showed that three bands of 726 bp, 516 bp, and 210 bp appeared in all three goat breeds, indicating three genotypes: heterozygous mutant GA (726 bp, 516 bp, and 210 bp), homozygous mutant AA (716 bp), and wild homozygous GG (516 bp, 210 bp). The enzyme digestion results are shown in Table 1. Figure 3 .

[0196] As for the other four mutation sites discovered, since there is no restriction endonuclease directly targeting them, the PCR-HRM method was used to analyze the other mutation sites.

[0197] 2.3.2 PCR-HRM analysis of the NDRG4 gene

[0198] Sequencing results revealed that the NDRG4 gene (GenBank accession number 102176452) harbored a single-base mutation in intron 3, as well as another in the ENON17 region and two in the 3'UTR. The mutations occurred at bp 27640465 on chromosome 18 of the NDRG4 gene, resulting in a G-to-A mutation (designated g. 27640465 G>A mutation); a T-to-C mutation (designated g. 27640826 T>C mutation) at bp 27640826 on chromosome 18 of the NDRG4 gene; and a C-to-G mutation (designated g. 27642312 C>G mutation) at bp 27642312 on chromosome 18 of the NDRG4 gene.

[0199] For the three mutation sites g. 27640465 G>A, g. 27640826 T>C, and g. 27642312 C>G, primers N17-1 (SNP2), i.e., the sequences shown in SEQ ID NOs. 9-10, N17-2 (SNP3), i.e., the sequences shown in SEQ ID NOs. 11-12, and N17-3 (SNP4), i.e., the sequences shown in SEQ ID NOs. 13-14, were designed (for specific primer sequence information, see Table 2 ). High-resolution melting curve analysis was used to genotype these three SNPs (e.g., Figures 4-6 ), it was found that these three mutation sites had three genotypes in the three goat populations, showing polymorphism.

[0200] PCR amplification products of different genotypes were sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The results showed that, compared with the AA type, the amplified sequence of the GG type in the G>A site at g. 27640465 G>A site had a G→A mutation at 229bp (calculated with the first position of the coding sequence as +1) in the NDRG4 gene. Compared with the CC type, the amplified sequence of the TT type in the T>C site at g. 27640826 T>C site had a T→C mutation at 209bp (calculated with the first position of the coding sequence as +1) in the NDRG4 gene. Compared with the GG type, the amplified sequence of the CC type in the C>G site at g. 27642312 C>G site had a C→G mutation at 283bp (calculated with the first position of the coding sequence as +1) in the NDRG4 gene. Figure 7 .

[0201] 2.3.3 Analysis of genetic parameters of SNPs in the NDRG4 gene

[0202] Genetic heterozygosity (He), genetic homozygosity (Ho), polymorphic information content (PIC), and effective allele number (Ne) are important parameters for evaluating population genetic variation. Different genetic parameters represent the essential genetic differences between populations. The genetic parameters of NDRG4 SNPs in Leizhou goats, Sichuan Black goats, and Nubian goats are shown in Tables 8 and 9.

[0203] Tables 8 and 9 show that the polymorphic information content (PIC) at the four variant loci for the three goat breeds ranged from 0.25 to 0.5, indicating moderate polymorphism and a relatively rich genetic profile. Furthermore, the homozygosity and heterozygosity at the g. 27640465 G>A and g. 27640826 T>C loci for the three goat breeds were similar, both around 0.50. Furthermore, the number of effective alleles was similar to the number of detected alleles, indicating that the alleles at these two variant loci are evenly distributed across the three goat populations. Meanwhile, the Nubian goat had higher Ho values ​​and lower He values ​​at the g. 27632588 A>G and g. 27642312 C>G loci, as well as the Sichuan Black goat's g. 27632588 A>G loci, indicating relatively stable genetic performance at these loci, making them suitable for seed preservation and enhancing heterosis in breeding efforts.

[0204] Chi-square tests showed that the g. 27640826 T>C locus was in Hardy-Weinberg equilibrium across all three goat populations, with extremely significant differences observed in Leizhou and Chuanzhong Black goats (P<0.01), and significant differences observed in Nubian goats (P<0.05). This suggests that all three populations are in a state of non-natural equilibrium, possibly due to long-term artificial selection for a trait associated with the g. 27640826 T>C locus. Meanwhile, all three goat breeds were in Hardy-Weinberg equilibrium at the g. 27642312 C>G locus, with no significant differences (P>0.05). This suggests that the g. 27642312 C>G locus may not be affected by current breeding practices, and that genetic changes at this locus remain random during the breeding process. At the g. 27640465 G>A locus, a deviation from Hardy-Weinberg equilibrium was observed in Chuanzhong Black goats, with extremely significant differences (P<0.01). However, this site showed Hardy-Weinberg equilibrium in Leizhou goats and Nubian goats (P>0.05), indicating that traits associated with the g. 27640465G>a site have undergone long-term artificial selection in Sichuan black goats, while the selection of these traits in Leizhou goats and Nubian goats was not obvious or was ignored.

[0205] Table 8. Genetic parameter analysis of the 27632588 A>G locus.

[0206]

[0207] Note: The numbers in parentheses indicate the number of individuals with this genotype. * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01); no mark indicates no significant difference (P > 0.05). PIC > 0.5 is highly polymorphic; 0.25 < PIC < 0.5 is moderately polymorphic; PIC < 0.25 is lowly polymorphic. LZ is Leizhou goat, CZ is Chuanzhong black goat; NS is Nubian goat. This annotation also applies to Tables 8, 9, 10 and 11.

[0208] Table 9 Genetic parameter analysis of the g. 27640465 G>A locus

[0209]

[0210] Table 10 Genetic parameter analysis of the g. 27640826 T>C locus

[0211]

[0212] Table 11 Genetic parameter analysis of the g. 27642312 C>G locus

[0213]

[0214] 2.3.4 Association analysis of SNPs of the NDRG4 gene with goat body size traits

[0215] The results of the association analysis of SNPs of the NDRG4 gene with the body size traits of Leizhou goats, Chuanzhong black goats and Nubian goats are shown in Tables 12 - 14.

[0216] Table 12 shows that at the g.27632588 A>G locus in the Leizhou goat population, the average chest and abdominal circumferences of GG individuals were significantly higher (p < 0.01), reaching 79.71 cm and 87.56 cm, respectively, compared to 76.25 and 83.57 cm for AA individuals. Furthermore, the average leg circumference of GG individuals was significantly higher (p < 0.05), reaching 7.4 cm, compared to 7.06 cm for AA individuals. Furthermore, the average height of AG individuals at this locus reached 54.93 cm, significantly higher than the 53.74 cm for AA individuals (p < 0.05). At the g.27640465 G>A locus, the average hind leg circumference of AA individuals was 27.18 cm, significantly higher than that of GG (24.47 cm) and GA (25.39 cm) individuals (p < 0.01 and p < 0.05, respectively). Moreover, the average height of GA type individuals at this location was significantly higher than that of GG type at 53.89 cm (p < 0.05), reaching 54.94 cm.

[0217] The results in Table 13 show that within the Sichuan Black Goat population, individuals with the TT genotype at the g. 27640826 T>C locus exhibited significantly lower average body length, body length, and chest circumference than the other two genotypes. In terms of body length, the average levels of CC and TC genotypes reached 73.48 cm and 72.84 cm, respectively, significantly exceeding the TT genotype's 69.30 cm (p < 0.01). In terms of body length, the average levels of CC and TC genotypes reached 70.37 cm and 69.70 cm, respectively, significantly exceeding the TT genotype's 66.36 cm (p < 0.01). In terms of chest circumference, the average level of TC genotypes reached 86.80 cm, significantly exceeding the TT genotype's 85.07 cm (p < 0.05). At the g. 27642312 C>G locus, the oblique body length and straight body length of CC type individuals reached 70.82 cm and 67.82 cm respectively, while the oblique body length of CG type and GG type individuals were significantly higher than that of CC type (p < 0.01), reaching 72.70 cm and 73.31 cm; at the same time, the straight body length of CG type and GG type individuals was also significantly higher than that of CC type (p < 0.01), reaching 69.59 cm and 70.07 cm.

[0218] The results in Table 14 show that in the Nubian goat population, individuals with the AG mutation at the g. 27632588 A>G locus had an average chest circumference of 83.81 cm and an average abdominal circumference of 88.59 cm. These AG individuals had significantly higher chest and abdominal circumferences than those with the AA mutation at 80.85 cm (p < 0.05) and 84.43 cm (p < 0.01). At the g. 27640465 G>A locus, individuals with the GA mutation had a significantly higher average body length of 67.19 cm than those with the GG mutation (p < 0.05). The average chest circumference and canthus circumference of individuals with the GA mutation were significantly higher than those with the AA mutation (p < 0.05), reaching 83.00 cm and 8.33 cm, respectively. The average rump width of individuals with the GG mutation was significantly higher than that of the AA mutation (p < 0.05), reaching 15.92 cm. At the g. 27640826 T>C locus, CC individuals had an average height of 71.40 cm, significantly greater than TC (66.46 cm) and TT (66.53 cm) individuals (p < 0.01). TT individuals also had an average rump width of 15.95 cm (15.95 cm) significantly greater than CC individuals (14.58 cm) (p < 0.05). At the g. 27642312 C>G locus, CC individuals had an average chest circumference of 85.27 cm, significantly greater than CG (81.42 cm) and GG (81.17 cm) individuals (p < 0.05).

[0219] Table 12 Association analysis between NDRG4 gene SNPs and body size traits in Leizhou goats

[0220]

[0221] Table 13 Association analysis between NDRG4 gene SNPs and body size traits in Sichuan Black Goats

[0222]

[0223] Table 14 Association analysis between NDRG4 gene SNPs and body size traits in Nubian goats

[0224]

[0225] 2.3.4 Association analysis between SNPs in the NDRG4 gene and goat litter size

[0226] The results of association analysis between SNPs of the NDRG4 gene and litter size in Leizhou goats, Sichuan black goats, and Nubian goats are shown in Tables 15-17.

[0227] Table 15 shows that in the Leizhou goat population, at the g. 27632588 A>G locus, AG individuals had significantly higher litter sizes (1.60, 1.78, and 1.66) in the first and second parities, respectively. These numbers were significantly higher than those of AA individuals in the first and second parities (p < 0.01), and significantly higher than those of AA individuals in the second parity (p < 0.05). At the g. 27640465 G>A locus, GA individuals had significantly higher litter sizes (1.89) in the third parity, compared to AA individuals (1.17) (p < 0.05). Furthermore, GG individuals had a significantly higher average litter size (1.65) than AA individuals (p < 0.05).

[0228] The results in Table 16 showed that in the Sichuan black goat population, among g. 27640826 T>C, the number of lambs born per litter of the TT genotype reached 2.10, which was significantly higher than the 1.90 of the CC genotype (p < 0.05).

[0229] The results in Table 17 show that in the Nubian goat population, g. 27632588 A>G, the number of lambs born per litter for AA and AG type individuals reached 1.79 and 1.81, respectively, which were significantly higher than the 1.29 for GG type (p < 0.05).

[0230] Table 15 Association analysis between NDRG4 gene SNPs and litter size in Leizhou goats

[0231]

[0232] Table 16 Association analysis between SNPs in the NDRG4 gene and litter size in Sichuan black goats

[0233]

[0234] Table 17 Association analysis between Nubian goat NDRG4 gene SNPs and goat litter size

[0235]

[0236] 2.4 Linkage disequilibrium analysis

[0237] Ardlie et al. (Ardlie KG, Kruglyak L, Seielstad M. Patterns of linkage disequilibrium in the human genome [J]. Nature Reviews Genetics, 2002, 3(4): 299-309.) proposed a threshold value of r^2 (a measure of linkage disequilibrium) to evaluate the strength of linkage disequilibrium between genetic loci. According to the findings, if the r^2 value is greater than 0.33, it can be considered to indicate that there is strong linkage disequilibrium between the loci. In addition, the closer the r^2 value is to 1, the stronger the linkage between the genetic loci. The analysis results of linkage disequilibrium are as follows: Figure 8 The results showed that there was no linkage disequilibrium at the four SNP loci in Leizhou goats and Sichuan black goats. In Nubian goats, the r values ​​of g. 27632588 A>G and g. 27640465 G>A, g. 27632588 A>G and g. 27640826 T>C, g.27632588 A>G and g. 27642312 C>G, g. 27640465 G>A and g. 27640826 T>C, g. 27640465 G>A and g. 27642312 C>G, and g. 27640826 T>C and g. 27642312 C>G were significantly higher than those of g. 2 >0.33, indicating strong linkage disequilibrium among all four SNPs in Nubian goats. Therefore, the present invention analyzed the effects of the combined genotypes of each of the four SNPs on growth traits and litter size in Nubian goats (as shown in Table 18).

[0238] The results showed that in the combined gene analysis of the g. 27632588 A>G and g. 27640465 G>A loci, regarding the body size traits of Nubian goats, it was found that the AGGA type was significantly longer than the AGGG type in body oblique length and straight length (P < 0.05), and extremely significantly higher than the AAAA type in chest circumference and abdominal circumference (P < 0.01); in addition, the AGGG type was significantly higher than the AAAA type in rump width (P < 0.05).

[0239] Combined genetic analysis of the g. 27632588 A>G and g. 27640826 T>C loci revealed that the AGTT genotype had significantly higher chest and abdominal circumferences than the AATC and AATT genotypes (P < 0.05). Furthermore, the AGTT genotype had a significantly higher rump width than the AATC genotype (P < 0.01) and significantly higher than the AATT, AGCC, AGTC, and GGTT genotypes (P < 0.05). Furthermore, the AATT genotype had a significantly higher hindquarter circumference than the AGTC genotype (P < 0.05). Regarding the reproductive trait of parity 1, the GGTT genotype had the lowest litter size, with the GGTT genotype having a significantly lower litter size than the AATT genotype (P < 0.01) and significantly lower than the AGCC, AGTC, and AGTT genotypes (P < 0.05).

[0240] In the combined gene analysis of the g. 27632588 A>G and g. 27642312 C>G loci, regarding the body size traits of Nubian goats, it was found that in terms of body oblique length, the GGGG type was significantly higher than the AAGG type (P < 0.05); in terms of chest circumference, the AGCG type was extremely significantly higher than the AACG type and AAGG type (P < 0.01), and significantly higher than the AACC type, AGGG type, and GGGG type (P < 0.05); in terms of abdominal circumference, the AGCG type was also extremely significantly higher than the AAGG type (P < 0.01), while the AAGG type was significantly lower than the AGCC type and AGGG type (P < 0.05); in terms of hind leg circumference, the AAGG type was significantly higher than the AACC type and AGCC type (P < 0.05).

[0241] In the combined gene analysis of the g. 27640465 G>A and g. 27640826 T>C loci, regarding the body size traits of Nubian goats, it was found that in terms of body height, the GGTC type was significantly higher than the GGTT type and the AATC type (P < 0.05); the GATC type was significantly higher than the AATC type in terms of body length, chest circumference and abdominal circumference (P < 0.05); in terms of hind leg circumference, the GATT type was extremely significantly higher than the GATC type (P < 0.01), and significantly higher than the AATC type, GGTC type and GGTT type (P < 0.05); in terms of canthus circumference, the GATT type was significantly higher than the AATC type and AATT type (P < 0.05).

[0242] In the combined gene analysis of the g. 27640465 G>A and g. 27642312 C>G loci, regarding the body size traits of Nubian goats, it was found that the AAGG type was significantly lower than the GACG type and GAGG type in both oblique and straight body length (P < 0.05), and extremely significantly lower than the AACC type and GACG type in chest circumference (P < 0.01). At the same time, the AACC type was extremely significantly higher than the AACG type and GGCG type in chest circumference (P < 0.01). In terms of abdominal circumference, the GACG type was significantly higher than the AACG and AAGG types (P < 0.05); in terms of hind leg circumference, the AAGG type was extremely significantly higher than the AACG and GGCG types (P < 0.01), and significantly higher than the GACC and GACG types (P < 0.05); in terms of canal circumference, the GACG type was extremely significantly higher than the AACG type (P < 0.01), and significantly higher than the GACC and GGCG types (P < 0.05); in terms of rump width, the AACG type was significantly lower than the AAGG, CACG, GGCG and GGGG types (P < 0.05).

[0243] In the combined gene analysis of the g. 27640826 T>C and g. 27642312 C>G loci, regarding body size traits, it was found that in terms of chest circumference, the TTCG type was significantly lower than the TCCC type and TTCC type (P < 0.05); in terms of abdominal circumference, the TCCG type was significantly higher than the TCGG type (P < 0.05); in terms of hind leg circumference, the TTCG type and TTGG type were both extremely significantly higher than the TCCG type (P < 0.01), and significantly higher than the TTCC type (P < 0.05).

[0244] Table 18 Effects of the combined genotypes of the four SNP loci on body size traits and litter size in Nubian goats

[0245]

[0246]

[0247]

[0248]

[0249] Note: The combined base types with less than 3 individuals are not listed in the table. The numbers in brackets represent the sample size.

[0250] 3 Discussion

[0251] 3.1 Analysis of genetic parameters of candidate gene polymorphic sites

[0252] The richness of genetic diversity is an important indicator of the genetic potential of a population. This richness is typically measured by the number of available alleles and the polymorphism information content (PIC). PIC, genetic homozygosity (Ho), genetic heterozygosity (He), effective number of alleles (Ne), gene frequency, and genotype frequency are important parameters for evaluating population genetic variation. Each of these genetic parameters represents the underlying genetic differences between populations. While Hardy-Weinberg equilibrium generally applies to randomly mating populations, various factors in real-world production can disrupt this equilibrium. Factors such as selective mating, artificial selection, natural selection, inbreeding, population subdivision, and other evolutionary forces can cause genotype frequencies in a population to deviate from those expected based on Hardy-Weinberg equilibrium. In other words, observed genotype frequencies may not conform to those predicted based on random mating and Mendelian inheritance.

[0253] In this study, the four SNPs (g. 27632588 A>G, g. 27640465 G>A, g. 27640826 T>C, and g. 27642312 C>G) showed moderate polymorphism (0.25 ≤ PIC ≤ 0.5) across all three goat breeds. In terms of polymorphic information content, a higher PIC value indicates greater genetic diversity and higher selection potential. This suggests that these four loci have high genetic diversity and good genetic stability, and are less susceptible to the influence of invasive goat breeds during long-term evolutionary selection.

[0254] Genetic homozygosity (Ho) and genetic heterozygosity (He) are used in genetic research to assess the degree of genotypic variation within a population or individual. Within a population, the closer the Ho and He values ​​are, the more likely the genotype at that locus is to be homozygous; the farther the values ​​are from each other, the more likely the genotype at that locus is to be heterozygous. This study found that the homozygosity and heterozygosity at the g.27640465 G>A locus and the g.27640826 T>C locus in three goat breeds were similar, both around 0.50, indicating that the alleles at these two loci are relatively evenly distributed across the three goat populations. Furthermore, at the g.27632588 A>G locus, the three breeds exhibited relatively high Ho values ​​and relatively low He values ​​compared to the two previously described loci. This suggests that the genetic performance at this locus is relatively stable, making it easier to preserve seeds and enhance heterosis in breeding efforts. At g.27642312 C>G, it can be found that the Ho value in the Nubian goat population is relatively high, while the Ho and He values ​​in the Sichuan Black Goat and Leizhou Goat populations are close to 0.50, indicating that this site may have undergone a certain degree of selective breeding in the Nubian goat population.

[0255] The effective allele number is the inverse of genetic homozygosity. The more evenly alleles are distributed within a population, the closer the effective allele number is to the absolute number of alleles detected. However, it is more directly related to the overall frequency distribution of alleles in the population. The concept of effective alleles is a measure of genetic diversity within a population, taking into account both the number and frequency of alleles. The Ne values ​​of the four SNPs in NDRG4 are all close to 2, which is close to the actual number of alleles detected, indicating that the two alleles at these loci are relatively evenly distributed within the population.

[0256] Chi-square tests showed that the g. 27640826 T>C locus exhibited deviations from Hardy-Weinberg equilibrium in all three goat breeds, with significant differences observed in Leizhou and Chuanzhong Black goats (P<0.01), and in Nubian goats (P<0.05). This suggests that all three populations exhibited a non-natural equilibrium, possibly due to long-term conscious breeding for traits associated with the g. 27640826 T>C locus. In contrast, at the g. 27642312 C>G locus, all three goat breeds achieved Hardy-Weinberg equilibrium, with no significant differences (P>0.05). This suggests that the g. 27642312 C>G locus may not be significantly affected by current breeding practices, and genetic variation at this locus may be more likely to occur randomly. At the g. 27640465 G>A locus, Chuanzhong Black goats exhibited a significant deviation from Hardy-Weinberg equilibrium (P<0.01). However, Leizhou and Nubian goats exhibited Hardy-Weinberg equilibrium at this locus (P>0.05), suggesting that traits associated with the G. 27640465 G>A locus may have undergone long-term artificial selection in Sichuan black goats, while selection for these traits may have been less pronounced or neglected in Leizhou and Nubian goats. A similar situation occurred at the g. 27632588 A>G locus in Nubian goats, where the difference was significant (P<0.05). However, Leizhou and Sichuan black goats exhibited Hardy-Weinberg equilibrium at this locus (P>0.05), suggesting that traits associated with the G. 27640465 G>A locus may have also undergone some long-term artificial selection in Nubian goats, while selection for these traits may have been less pronounced or neglected in Leizhou and Sichuan black goats.

[0257] 3.2 Analysis of NDRG4 gene polymorphism

[0258] In the present invention, four mutations, namely g. 27632588A>G, g. 27640465 G>A, g. 27640826 T>C and g. 27642312 C>G, exist in the three breeds of Leizhou goat, Sichuan black goat and Nubian goat.

[0259] The g. 27632588 A>G mutation is located in the intron 3 region of the NDRG4 gene and has three genotypes (AA, GA, and GG). Association analysis revealed that in the Leizhou goat population, the average chest and abdominal circumferences of GG-type individuals were significantly higher (p < 0.01) than those of AA-type individuals (76.25 and 83.57 cm), reaching 79.71 cm and 87.56 cm, respectively. Furthermore, the average canine circumference of GG-type individuals was significantly higher (p < 0.05) than that of AA-type individuals (7.06 cm), reaching 7.4 cm. Furthermore, the average height of AG-type individuals at this locus reached 54.93 cm, significantly higher than the 53.74 cm of AA-type individuals (p < 0.05). In the Nubian goat population, the average chest circumference of individuals with the AG genotype reached 83.81 cm and the average abdominal circumference reached 88.59 cm, which were significantly higher than those of individuals with the AA genotype (80.85 cm (p < 0.05) and 84.43 cm (p < 0.01). This suggests that the g. 27632588 A>G locus may be a key gene influencing skeletal development in both Leizhou and Nubian goats. In body size traits, the G gene is dominant in both Leizhou and Nubian goats. However, in Sichuan Black goats, no significant differences in body size were observed between individuals with different genotypes at this mutation (P > 0.05).

[0260] In terms of reproductive traits, individuals with the g. 27632588 A>G locus in the Leizhou goat population had significantly higher numbers of lambs born in the first and second parities (1.60, 1.78, and 1.66), respectively. These numbers were significantly higher than those of the AA locus in the first and second parities (p < 0.01), and significantly higher than those of the AA locus in the second parity (p < 0.05). In the Nubian goat population, individuals with the AA and AG loci at this locus had significantly higher numbers of lambs born in the first parity (1.79 and 1.81), respectively, both significantly higher than the 1.29 born by the GG locus (p < 0.05). This suggests that g. 27632588 A>G may also be a key gene influencing reproductive traits in both the Leizhou and Nubian goats, with AG being the dominant genotype. Therefore, the g. 27632588 A>G locus could serve as a candidate locus for selection in breeding for both reproductive traits and body size.

[0261] g. 27640465 G>A is a synonymous mutation located in exon 17 of the NDRG4 gene. In terms of body measurements, the average hind leg circumference of individuals with the AA genotype at this locus in the Leizhou goat population is 27.18 cm, significantly higher than that of individuals with the GG (24.47 cm) and GA (25.39 cm) genotypes (p < 0.01 and p < 0.05). Furthermore, the average height of individuals with the GA genotype at this locus is significantly higher (p < 0.05), reaching 54.94 cm, compared to 53.89 cm for individuals with the GG genotype. In the Nubian goat population, the average body length of individuals with the GA genotype is significantly higher (p < 0.05), reaching 67.19 cm. The average chest circumference and canthus circumference of individuals with the GA genotype are significantly higher (p < 0.05), reaching 83.00 cm and 8.33 cm, respectively, than those with the AA genotype. The average rump width of GG individuals was significantly higher than that of AA individuals (p < 0.05), reaching 15.92 cm. This suggests that the g. 27640465 G>A locus may be a key gene influencing the excellent body size traits of Leizhou and Nubian goats, with A being the dominant gene in Leizhou goats and GA being the dominant genotype in Nubian goats.

[0262] Regarding reproductive traits, the GA type at the g. 27640465 G>A locus in the Leizhou goat population had a significantly higher number of lambs born per three litters (1.89) than the AA type (1.17) (p < 0.05). Furthermore, the GG type had a significantly higher average number of lambs (1.65) than the AA type (p < 0.05). This suggests that the g. 27640465 G>A locus may also be a key gene influencing the reproductive traits of the Leizhou goat, with G being the dominant gene.

[0263] g. 27640826 T>C is a mutation in the 3'UTR region. In terms of body measurements, individuals with the TT genotype in the Sichuan Black Goat population showed significantly lower average body length, body length, and chest circumference than the other two genotypes. In terms of body length, the average levels of individuals with the CC and TC genotypes reached 73.48 cm and 72.84 cm, respectively, significantly higher than the TT genotype's 69.30 cm (p < 0.01). In terms of body length, the average levels of individuals with the CC and TC genotypes reached 70.37 cm and 69.70 cm, respectively, significantly higher than the TT genotype's 66.36 cm (p < 0.01). In terms of chest circumference, the average level of individuals with the TC genotype reached 86.80 cm, significantly higher than the TT genotype's 85.07 cm (p < 0.05). The average height of CC-type individuals in the Nubian goat population reached 71.40 cm, significantly greater than that of TC-type individuals (66.46 cm) and TT-type individuals (66.53 cm) (p < 0.01). The average hind leg circumference of TT-type individuals (31.89 cm) was significantly greater than that of TC-type individuals (30.10 cm) (p < 0.05). Furthermore, the average rump width of TT-type individuals (15.95 cm) was also significantly greater than that of CC-type individuals (14.58 cm) (p < 0.05). This suggests that TT is a disadvantageous genotype in the Sichuan Black Goat population. However, in the Nubian goat population, both the T and C alleles have advantages in body size traits.

[0264] In terms of reproductive traits, the TT genotype at the g. 27640826 T>C locus in the Sichuan Black Goat population had a litter size of 2.10 lambs, significantly higher than the 1.90 lambs of the CC genotype (p < 0.05), indicating that T is the dominant gene in the Sichuan Black Goat population.

[0265] g. 27642312 C>G is also a mutation in the 3'UTR region. Regarding body size, the oblique length of CG and GG individuals in the Sichuan Black Goat population was significantly higher (p < 0.01), reaching 72.70 cm and 73.31 cm, respectively, compared to the CC type (70.82 cm). Furthermore, the vertical length of CG and GG individuals was significantly higher (p < 0.01), reaching 69.59 cm and 70.07 cm, respectively, compared to the CC type (67.82 cm). However, the average chest circumference of CC individuals in the Nubian Goat population reached 85.27 cm, significantly greater than that of CG individuals (81.42 cm) and GG individuals (81.17 cm) (p < 0.05). This indicates that CC is a disadvantaged genotype in the Sichuan Black Goat population. However, CC is a dominant genotype in chest circumference in the Nubian Goat population. At the same time, in terms of reproductive traits, there was no significant difference among the genotypes of this site in the three goat groups.

[0266] 3.3 Linkage disequilibrium analysis

[0267] Genetic linkage, the phenomenon in which genes or genetic markers located close to each other on the same chromosome tend to be inherited together, is the basis for linkage disequilibrium analysis. Linkage disequilibrium occurs when the observed pattern of genetic linkage deviates from that expected under standard genetic models, suggesting potential genetic interactions or recombination events that may be influencing phenotypic traits. Analysis of linkage disequilibrium (LD) involves examining the distribution of genetic markers or alleles in a population and comparing it to the expected pattern based on genetic models. This analysis helps identify regions of the genome that are under selective pressure or participate in complex genetic interactions.

[0268] The results of this study showed that in Nubian goats, the AGGA genotype at the g. 27632588 A>G and g. 27640465 G>A loci combined had significantly higher chest and abdominal circumferences than the AAAA genotype (P<0.01). In single-locus analysis, the AG genotype also had significantly higher chest and abdominal circumferences than the AA genotype (P<0.05, P<0.01). At the g. 27640465 G>A locus, the average chest circumference of the GA genotype was significantly higher than that of the AA genotype (P<0.05). This suggests an additive effect at these two loci, meaning that the genotype combination at these two loci has an additive effect on body size traits such as chest circumference and abdominal circumference, resulting in a more significant impact.

[0269] Combination analysis of the g. 27632588 A>G and g. 27640826 T>C loci in Nubian goats revealed that the AGTT genotype had significantly higher chest and abdominal circumferences than the AATC and AATT genotypes (P < 0.05). Furthermore, the AGTT genotype had a significantly higher rump width than the AATC genotype (P < 0.01) and significantly higher than the AATT, AGCC, AGTC, and GGTT genotypes (P < 0.05). Furthermore, the AATT genotype had a significantly higher hindquarter circumference than the AGTC genotype (P < 0.05). Among reproductive traits, the GGTT genotype had the lowest litter size in parity 1, which was significantly lower than the AATT genotype (P < 0.01) and significantly lower than the AGCC, AGTC, and AGTT genotypes (P < 0.05). This indicates that the genotype combination at these two loci significantly affects both body size and reproductive traits. In single-locus analysis, the chest and abdominal circumferences of Nubian goats with the g. 27632588 A>G locus were significantly greater in AG-type Nubian goats than in AA-type individuals (P < 0.05, P < 0.01). Furthermore, the number of lambs per litter was significantly lower in GG-type Nubian goats than in AA and AG-type Nubian goats (P < 0.05). In the g. 27640826 T>C locus analysis, the average level of hind leg circumference in TT-type Nubian goats was significantly greater than that in TC-type Nubian goats (P < 0.05). Furthermore, the average level of rump width in TT-type Nubian goats was significantly greater than that in CC-type Nubian goats (P < 0.05). This suggests that the genotype at a single locus also has a certain influence on body size and reproductive traits. Thus, there is an additive effect between the g. 27632588 A>G and g. 27640826 T>C loci on both body size and reproductive traits. Among body size and reproductive traits, the AGTT genotype performed best in chest circumference, abdominal circumference, and rump width, while the GGTT genotype performed worst in reproductive traits. Therefore, the effects of these two loci on these traits may be the most significant, and the AGTT genotype may be the dominant combination genotype.

[0270] Combined genetic analysis of the g. 27632588 A>G and g. 27642312 C>G loci in Nubian goats revealed that, for chest circumference, the AGCG genotype was significantly higher than the AACG and AAGG genotypes (P < 0.01), and significantly higher than the AACC, AGGG, and GGGG genotypes (P < 0.05). For abdominal circumference, the AGCG genotype was also significantly higher than the AAGG genotype (P < 0.01), while the AGCC and AGGG genotypes were also significantly higher than the AAGG genotype (P < 0.05). In single-locus analysis, the chest and abdominal circumference of AG-type Nubian goats at the g. 27632588 A>G locus were significantly higher than those of AA-type individuals (P < 0.05, P < 0.01). At the g. 27642312 C>G locus, CC-type individuals had significantly larger chest circumference than both CG-type and GG-type individuals (P < 0.05). This indicates that the genotypes at these two loci have an additive effect on the body size traits of chest circumference and abdominal circumference, with the combined effect being greater than that of a single locus, consistent with an additive effect. Furthermore, the significant dominance of the AGCG genotype in chest circumference and abdominal circumference suggests that this combined genotype may have the greatest impact on these two traits, while the significant dominance of the CC genotype in chest circumference suggests that this genotype may be the dominant genotype at this locus.

[0271] Combined genetic analysis of the g. 27640465 G>A and g. 27640826 T>C loci in Nubian goats revealed that the GGTC genotype exhibited significantly higher body height than the GGTT and AATC genotypes (P < 0.05). In terms of chest circumference, the GATC genotype exhibited significantly higher chest circumference than the AATC genotype (P < 0.05). In terms of hindquarter circumference, the GATT genotype exhibited significantly higher hindquarter circumference than the GATC genotype (P < 0.01) and significantly higher hindquarter circumference than the AATC, GGTC, and GGTT genotypes (P < 0.05). In terms of canthus circumference, the GATT genotype exhibited significantly higher canthus circumference than the AATC and AATT genotypes (P < 0.05). In single-locus analysis, at the g. 27640465 G>A locus, the average level of body slant length in GA genotype individuals was significantly higher than that in GG genotype individuals (P < 0.05). Furthermore, the average levels of chest circumference and canthus circumference in GA genotype individuals were significantly higher than those in AA genotype individuals (P < 0.05). The average level of rump width of GG type individuals was significantly higher than that of AA type individuals (p < 0.05).

[0272] At the g. 27640826 T>C locus, CC-type individuals showed significantly greater height than TC-type and TT-type individuals (p < 0.01). Furthermore, TT-type individuals had significantly greater hind leg circumference than TC-type individuals (p < 0.05). Furthermore, the average level of rump width in TT-type individuals was significantly greater than that in CC-type individuals (p < 0.05). These results suggest that these two loci may have an additive effect on body size traits in goats.

[0273] Combined genetic analysis of the g. 27640465 G>A and g. 27642312 C>G loci in Nubian goats revealed that for body size traits, the AACC and GACG genotypes were significantly higher than the AAGG genotype (P<0.01). Furthermore, the AACC genotype was significantly higher than the AACG and GGCG genotypes (P<0.01). For canine circumference, the GACG genotype was significantly higher than the AACG genotype (P<0.01) and significantly higher than the GACC and GGCG genotypes (P<0.05). For rump width, the GGCG and GGGG genotypes were significantly higher than the AACG genotype (P<0.05). Single-locus analysis revealed that at the g. 27640465 G>A locus, the average levels of chest and canine circumference in GA genotypes were significantly higher than those in AA genotypes (P<0.05). The average level of rump width in GG genotypes was significantly higher than that in AA genotypes (P<0.05). At the g. 27642312C>G locus, CC individuals had significantly larger chest circumference than both CG and GG individuals (p < 0.05). These results suggest an additive effect between these two loci, with the most significant effect on chest circumference. Furthermore, AACC individuals exhibited an advantage in chest circumference, potentially representing a dominant genotype.

[0274] Combined genetic analysis of the g. 27640826 T>C and g. 27642312 C>G loci in Nubian goats revealed that for chest circumference, the TCCC and TTCC types were significantly higher than the TTCG type (P < 0.05). For hind leg circumference, the TTCG and TTGG types were both extremely significantly higher than the TCCG type (P < 0.01) and significantly higher than the TTCC type (P < 0.05). Analysis of individual loci revealed that at the g. 27640826 T>C locus, the TT type had significantly higher hind leg circumference than the TC type (P < 0.05). At the g. 27642312 C>G locus, the CC type had significantly larger chest circumference than both the CG and GG types (P < 0.05). These results suggest an additive effect between these two loci.

[0275] 4. Conclusion

[0276] (1) g. 27632588 A>G locus can be used as a molecular marker to improve the chest circumference, abdominal circumference, canine circumference and body height of Leizhou goats; it can also be used as a molecular marker to increase the chest circumference and abdominal circumference of Nubian goats. At the same time, this locus has a significant effect on the number of lambs born in both Leizhou goats and Nubian goats (p < 0.05), indicating that this locus can be used as a molecular marker to affect the reproductive capacity of these two goat breeds.

[0277] (2) The g. 27640465 G>A locus can be used as a molecular marker to improve the hind leg circumference of Leizhou goats; it can be used as a molecular marker to improve the body length, chest circumference, tube circumference and rump width of Nubian goats; at the same time, this locus has a significant effect on the number of lambs born in Leizhou goats (p < 0.05), and can be used as a molecular marker to affect the reproductive capacity of the Leizhou goat breed.

[0278] (3) g. The 27640826 T>C locus can be used as a molecular marker to improve the oblique length, straight length and chest circumference of Sichuan black goats; it can be used as a molecular marker to increase the body height, hind leg circumference and rump width of Nubian goats; at the same time, this locus has a significant effect on the number of lambs born in Sichuan black goats (p < 0.05), and can be used as a molecular marker to affect the reproductive capacity of Sichuan black goats.

[0279] (4) g. 27642312 C>G can be used as a molecular marker to improve the oblique length and straight length of the body of Sichuan Black Goats; it can also be used as a molecular marker to increase the chest circumference of Nubian Goats;

[0280] (5) AGGA type (g. 27632588 A>G and g. 27640465 G>A), AGTT type and AATT type (g.27632588 A>G and g. 27640826 T>C), AGCG type (g. 27632588 A>G and g. 27642312 C>G), GGTC type and GATT type and GATC type (g. 27640465 G>A and g. 27640826 T>C), AACC type and GACG type and GGCG type (g. 27640465 G>A and g. 27642312 C>G), TCCC type and TTCG type and TTGG type (g. 27640826 T>C and g. 27642312 C>G) is the best combination genotype for breeding excellent body size traits of Nubian goats.

[0281] (6) The combined genotype GGTT (g. 27632588 A>G and g.27640826 T>C) should be avoided when breeding Nubian goats for reproductive traits.

[0282] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of a method for detecting a SNP locus genotype combination material associated with growth traits of Nubian goats in screening Nubian goats with high growth traits, characterized in that: The SNP site genotype combination includes one or more of A1 to A6, and the genotypes corresponding to the SNP site genotype combination shown in A1 to A6 are B1 to B6 respectively; A1 is g.27632588A>G and g.27640465G>A; A2 is g.27632588A>G and g.27640826T>C; A3 is g.27632588A>G and g.27642312C>G; A4 has g.27640465G>A and g.27640826T>C; A5 is g.27640465G>A and g.27642312C>G; A6 has g.27640826T>C and g.27642312C>G; The g.27632588A>G means that the base at position 27632588 on the sheep chromosome 18 is A / G, the g.27640465G>A means that the base at position 27640465 on the sheep chromosome 18 is G / A, the g.27640826T>C means that the base at position 27640826 on the sheep chromosome 18 is T / C, and the g.27642312C>G means that the base at position 27642312 on the sheep chromosome 18 is C / G. The SNP site is located on the NDRG4 gene, and the GenBank accession number of the NDRG4 gene is 102176452; The genotype of the B1 is AGGA, the genotype of the B2 is one of AGTT and AATT, the genotype of the B3 is AGCG, the genotype of the B4 is one of GGTC, GATT and GATC, the genotype of the B5 is one of AACC, GACG and GGCG, and the genotype of the B6 is one of TCCC, TTCG and TTGG; Among them, sheep with any of the genotypes B1 to B6 are Nubian goats with high growth traits.

2. The use according to claim 1, characterized in that The substance is a PCR primer and / or sequencing primer designed for the SNP site to be detected.

3. The use according to claim 1, characterized in that The substance is a primer for detecting g.27632588A>G as shown in SEQ ID NO.1 to SEQ ID NO.2; The substance is a primer for detecting g.27640465G>A as shown in SEQ ID NO.9 to SEQ ID NO.10; The substance is a primer for detecting g.27640826T>C as shown in SEQ ID NO.11 to SEQ ID NO.12; The substance is a primer for detecting g.27642312C>G as shown in SEQ ID NO.13 to SEQ ID NO.

14.

4. A method for improving the growth traits of Nubian goats, characterized in that: The following steps are involved: Detect the nucleotides of each SNP site in the SNP site genotype combination described in claim 1 in the genome of the sheep to be tested, and determine the genotype of the sheep to be tested corresponding to the SNP site genotype combination. If the genotype of the SNP site genotype combination of the sheep to be tested belongs to any one of B1 to B6 described in claim 1, the sheep to be tested is a Nubian goat with a high growth trait.

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