Analysis of the eighth intron polymorphism of NR6A1 gene in sheep and its correlation with lumbar spine number

Abstract For revealing molecular markers related to the traits of multiple lumbar vertebrae in sheep, we analyze the relationship between NR6A1 gene polymorphism and lumbar vertebrae number traits in Xinjiang Kazakh sheep. Lumbar muscle tissues were collected from 6-lumbar spine (L6) Kazak sheep and 7-lumbar spine (L7) Kazak sheep and the intron-8 of NR6A1 gene was amplified by PCR. The SNP locus was detected by the PCR-SSCP method. One-Way ANOVA and an Independent Chi-square Test is adopted to analyze the genotype association with lumbar spine number variation. There were two SNP loci in the intron-8 of the NR6A1 gene: IVS8-188 and IVS8-281. One-Way ANOVA and Independent Chi-square Test indicated a significant association between IVS8-281 and lumbar spine number. The SNP locus of NR6A1 gene intron 8 (IVS8-281G > A) could play a certain role in the variation of lumbar spine number in Xinjiang Kazakh sheep and demonstrates potential to accelerate the sheep breeding of selection process.


Background
Small ruminants, particularly native breeds kinds, play a significant role in the livelihoods of a considerable part of the human population in the tropics from socio-economic aspects. 1,2 Thus, combined trials with emphasis on administration and genetic progress to improve animal outputs are of decisive significance. [3][4][5] Economical and biological efficiency of sheep production enterprises generally improves by increasing productivity and reproductive performance of ewes. [6][7][8] The vertebrate spine consists of a series of repeating bones that extend from front to back along the axis of the body. It includes neck vertebrae, thoracic, lumbar, sacrum, and coccyx. Changes in the number of thoracic and lumbar vertebrae are observed in several mammals, such as the ancestors of commercial pigs in Europe, wild boars and native pigs, [9][10][11] Qinchuan cattle, 12 and Jinchuan yak. 13,14 Multiple vertebrae also is found in Mongolian sheep in East Wuzhu Muqin Banner and West Wuzhu Muqin Banner of Xilingol League in Inner Mongolia, 15 Small-Tailed Han sheep in Hebei, 16 Kazakh and Altai sheep in Xinjiang. 17 Studies have confirmed that carcass length increase by 2.4 cm or 3.5 cm for every additional thoracic or lumbar vertebra in Mongolian sheep. 18 Our previous study has found that carcass length increased by 2.22 cm, and carcass weight increased by 1.68 kg when adding a lumbar vertebra; while carcass length increased by 2.93 cm, and carcass weight increased by 1.9 kg when adding a thoracic vertebra in Kazakh sheep. 19 It indicates that the vertebra number is an economically important trait in livestock and it relevant to the weight and carcass length. Therefore, multi-vertebra is considered to have a great valueadded potential for sheep production.
Nuclear receptor subfamily 6, group A, member 1 (NR6A1), also known as germ cell nuclear factor (GCNF), 20 is located in the third chromosome of sheep, with a total length of 73,741 bp and a total of 9 introns. NR6A1 is highly expressed in mature germ cells, embryonic stem cells at the differentiation stage, and germ cell tumors such as teratomas, etc., with certain spatiotemporal expression specificity and tissue expression specificity. Until now, through a lot of research, the NR6A1 gene holds a significant part in cell differentiation, development, metabolism, and breed. 21 NR6A1 is related to the number of spines. 22 Studies on commercial breeds of Pigs in Europe and local breeds of pigs or wild boars in Asia reveal that mutations of the NR6A1 gene effect the change of the number of spines, 23 and it can also be applied as a marker of genetic differentiation for species classification. 24 The relationship between this gene and the multi-vertebra trait is also confirmed in Hebei Xiaotailhan sheep. 16 Increasing meat production using scientific, accurate, and precise selective programs are one of the most important goals for the genetic improvement of goats. This can be done by identifying the genotype of reproductive and productive traits of animals and their relationships, through determining the polymorphism and finding the phylogenetic relationships of domestic animals. [25][26][27] Determination of gene polymorphism is important in farm animals breeding [27][28][29] to define genotypes of animals and their associations with productive, reproductive, and economic traits. 30 On the other hand, a species without enough genetic diversity is thought to be unable to cope with changing environments or evolving competitors and parasites. 31 In addition, the ability of a population to respond adaptively to environmental changes depends on its level of genetic variability or diversity. 32 Thus, genetic diversity in indigenous breeds is a major concern considering the necessity of preserving what may be a precious and irreplaceable richness, regarding new productive demands. 8 Conservation should be based on a deep knowledge of the genetic resources of the specific breed. 33 Therefore, it is important to try to genetically characterize indigenous breeds 29 and the applications of molecular genetics have many important advantages. 34 Moreover, studies indicate that mutations hidden in introns may play a role in transcription and mRNA shearing. 35 And the epigenome comprising different mechanisms, e.g., DNA methylation, remodeling, histone tail modifications, chromatin microRNAs, and long non-coding RNAs, interact with environmental factors like nutrition, pathogens, climate to influence the expression profile of genes and the emergence of specific pheno-types. 36 Multi-level interactions between the genome, epigenome, and environmental factors might occur. Furthermore, numerous lines of evidence suggest the influence of epigenome variation on livestock production. 36,37 In this study, 6-lumbar spine Kazak sheep (n ¼ 58) and 7-lumbar spine Kazak sheep (n ¼ 59) are selected as experimental samples to assay the correlation between SNP of the intron-8 of NR6A1 gene and poly lumbar traits. Our results uncover molecular genetic markers related to sheep the multi-vertebra trait and will speed up the process of breeding mutton sheep breeds.

Samples
Xinjiang Kazakh sheep were selected as the experimental animals. Sampling from sheep slaughterhouse (Shihezi, Xinjiang, China) was divided into two groups: adult Kazakh sheep with six lumbar individual groups (n ¼ 58) and adult Kazakh sheep with seven lumbar individual groups (n ¼ 59). Collect the sheep's last pair of muscles near the lumbar vertebrae and stored them into the cryopreserved tubes without RNase in a refrigerator at À80 C. The vertebral characteristics and traits of Kazakh sheep have been described in the results of the team's previous studies. 19

DNA extraction
Complete genome DNA from sheep muscle was extracted following the protocol of TIANamp Genomic DNA Kit (DP304) (Beijing, China, Tiangen Biochemical Technology co., LTD) and preserved at À20 C. To assess the density and purity of the genome were used 1% agarose gel electrophoresis and the optical density and DNA concentration detected with the Nanodrop (ND1000) (Wilmington, DE, USA, Thermo Fischer Scientific).

PCR amplification and sequence
Primer 5.0 software was adopted to design primers for amplifying the intron-8 of NR6A1 (GenBank accession no. NC_019460.2). Primer sequences are added in Supplementary Table S1 (Rebo Hing Technology co., LTD, Beijing, China). DNA pools were constructed by mixing 20 genomic samples (2 lL/each sample) from 6-lumbar spine or 7-lumbar spine individuals, respectively. The DNA pool was carried out PCR of intron-8 amplification and screening SNP sites. Amplification was performed in an iCycler (Bio-Rad, Hercules, CA, USA). Each system comprised: 50 ng DNA, 10 lL Taq PCR Master Mix (2Â, with Blue Dye), 20 pmol forward and reverse primers, and double-distilled H 2 O to 20 lL. Successful PCR products were cloned according to Supplementary Table S2. After completion of the reaction, amplicons were electrophoresed in a 1% agarose gel (containing the ethidium bromide) to be compared with the 2000 bp DNA Ladder (Takara). Sequence the pooled PCR products by a specialized sequence company (Rebo Hing Technology Co., LTD, Beijing, China). The sequence results were analyzed using DNAMAN 7 software, showing two SNP sites (located at 188 bp, 281bp of the amplified fragment). PCR primers applied to amplify SNPs are reported in Supplementary Table S3. Except for the primers, the PCR amplification system for loci was the same as the intron-8. The standardized amplification conditions are shown in Supplementary Table S4 (Rebo Hing Technology co., LTD, Beijing, China). The amplification was verified by staining and electrophoresis with 2% agarose gel (containing the ethidium bromide), and the target band was identified according to 50 bp DNA Ladder (Takara).

SSCP analysis and sequence
The SSCP-genotyping reactions to NR6A1 gene SNP analysis were performed using SSCP analysis as described by Zhang et al. 20 and stained with silver staining as described by Byun et al. 38 PCR amplification loaded on 10% acrylamide: bisacrylamide (39:1) (Bio-Rad) gels in 1 Â TBE buffer and constant current 30 mA for 10-14 h after a pre-run at 300 V for 15 min. 38 Analyze the results of the SSCP and select the PCR product corresponding to the band type. The purified products were further sequenced using a commercial service (Rebo Hing Technology co., LTD, Beijing, China) to confirm the mutation location and base sequence. Finally, DNAMAN 7 software was used to complete sequence alignments and translations. 39 Genotyping and statistical analysis Based on genotype statistics, Genotypic and allelic frequencies were predicted. One-Way ANOVA and Independent Chi-square test was adopted to analyze the correlation between the NR6A1 mutation loci and the characteristics (lumbar spine number) of Kazakh sheep. Statistical analysis was performed on IBM SPSS Statistics 21.

PCR-SSCP and sequence analysis
The NR6A1 gene has nine introns. Polymorphisms were found in the intron-8 and intron-9 of the NR6A1 gene, and no polymorphism was found in introns 1-7. However, SNP sites in the ninth intron were relatively clustered, we choose to study the loci of the intron-8. To authenticated the NR6A1 intron-8 polymorphisms, three pairs of PCR primers were designed to amplify the intron-8. The sequence results of amplification products of intron 8 of the NR6A1 gene were analyzed by Chromas and DNAMAN 7 software. Two mutations were found, named IVS8-188 (located 188 bp of the amplified fragment) and IVS8-281 (located 281 bp of the amplified fragment), respectively (Figs. S1 and S2).
Sequence variability of NR6A1 with different numbers of lumbar vertebrae in 117 Kazakh sheep was further validated by PCR-SSCP and DNA sequence. For IVS8-281, there were three unique band patterns, namely three-band patterns and two-band patterns of two types. Pattern I and pattern II consisted of two bands named (AA) and (GG), and pattern III consisted of three bands named (AG) (Fig. 1). The PCR sequence results also recognized the presence of three genotypes (Fig. 2). The PCR-SSCP analysis of the IVS8-188 locus is shown in Supplementary Fig. S3.

Genetic polymorphism of intron-8 of NR6A1 gene in sheep
Allele and genotype frequencies of the different numbers of lumbar vertebrae sheep at locus IVS8-281 are shown in Table 1. The A allele frequencies in L6 and L7 phenotypes were 0.585 and 0.819, respectively, and the A allele at this SNP locus was the protogene. By calculating the Chi-square values of theoretical data AA and measured data of sheep with different genotypes, so the Kazakh sheep were not in Hardy-Weinberg Equilibrium (p > 0.05). For the group, the traits that may be controlled by genes have been artificially and unconsciously selected, and the genetic balance has been broken.

Association analysis of polymorphisms with quantitative traits of lumbar vertebrae
The results of the association between the different genotypes of the SNP locus and the number of lumbar vertebrae were based on an one-way ANOVA ( Table 2). The results indicated that the SNP locus was significantly associated with vertebrae number (p < 0.01). The results of the Chi-square independence test demonstrated significant differences between individuals of sheep lumbar 6 and lumbar 7 for the IVS8-281 locus (v 2 ¼ 24.46, df ¼ 2, p < 0.01). Therefore, the number of lumbar vertebrae was associated with gene polymorphism.

Discussion
The main purpose of animal reproduction is to enhance economic quality and fecundity. Meat productivity is an economically vital trait in cattle, which is determined by the growth character, the reproduction, the quality character of meat, and so on. We should start with the key genes and molecular genetic mechanism to study related traits, to obtain molecular means to improve the meat quality of domestic livestock. The phenomenon of multiple spines is a beneficial mutation existing in sheep. The lengthening of the spine of individuals with multiple spines can lead to an increase in body size and significantly improve carcass meat production. Therefore, the decipherment of the genetic mechanism of spine variation is a prerequisite to promote greater selective breeding of sheep. So far, several genes regulating the development of sheep spine have been revealed, including the Homeobox family genes, 40 Gdf11, 41 ActRIIB, 42 Btg2, 43 VRTN, 44 NR6A1, 16 and other genes. One of the important genes related to the quantitative traits of the lumbar spine is the NR6A1 gene. NR6A1 gene with SNP (748 C > T) is associated with a higher number of vertebrae in pigs. 45 SNP (T25G) on exon 5 of NR6A1 gene may affect the multi-vertebra character of Hebei Small tail Han sheep. 16 When the NR6A1 gene is knocked out, development of mouse embryos stop at celestial node 9.5, generating somites more than 12 fewer than that of the wild type. 46 To date, NR6A1 gene polymorphism in Kazakh sheep has been less studied. This work selected the NR6A1 gene as a target gene to screen the polymorphism locus of the lumbar spine in Kazakh sheep.
For the first time, we proposed a polymorphic site in the intron-8 of the NR6A1 gene, which was significantly related to the multiple lumbar traits of Kazakh sheep, providing a new basis for sheep breeding. In this study, we found two SNP sites in intron-8 through DNA-pool sequence. Two polymorphic loci   carry out PCR-SSCP analysis, and two SSCP patterns were found at IVS8-188 and three SSCP patterns were found at IVS8-281. The gene frequency and genotype frequency of this locus in sheep of two traits were calculated respectively, and the mutation of G < A at this locus was obtained, and A was the dominant allele. According to the One-Way ANOVA and Independent Chi-square Test, there were significant differences in IVS8-281 between 6 lumbar spines and 7 lumbar spine individuals in sheep (p < 0.01), that is, mutations in IVS8-281 of NR6A1 gene intron-8 is related to poly lumbar traits. Despite the mutation of the intron region of the NR6A1 gene does not cause amino acid changes, its polymorphism may regulate the gene expression. The mutation site may play a cis role, through mutation to change the high-level confirmation of nucleic acid molecules, thereby affecting the gene transcription, or affecting post-transcriptional intron splicing, and then affecting the protein function, which is associated with certain traits. NR6A1 is a transcription factor, and this mutation may affect the inhibition of NR6A1 transcription. This requires subsequent molecular biological testing of larger populations and the associated analysis of genetic polymorphisms with multiple vertebral traits. The carcass length and meat yields were counted in the early stage in Kazakh sheep with different spine numbers and the results demonstrated that the carcass length and weight of sheep with multiple spine number were increased. 19 For the investigation of the spines number of Qinchuan cattle and its hybrid cattle, it is also found phenomena of multiple spines, 12 which only accounted for 5.41% of the surveyed population. In the Jinchuan yak population, the number of vertebrate variation individuals is relatively large, accounting for the overall 52%. 14 Moreover, the multi-rib Jinchuan yak's reproductive rate, individual final calf number, and meat production capacity are better than the average individual. 13 We can also record the litter size in the process of progenitive multi-vertebral sheep for selecting better breeds.
We identified for the first time a novel genetic variant of intron-8 of the NR6A1 gene in Kazakh sheep by PCR-SSCP. SNP locus can be used as a motif in Kazakh sheep for early screening of sheep using molecular marker technology. Our results demonstrates potential to accelerate the sheep breeding of selection process in the future.

Conclusions
An SNP is identified in intron-8 of the sheep NR6A1 gene by PCR-SSCP analysis (IVS8-281: G > A). One-Way ANOVA and Independent Chi-square Test results demonstrates that the genotype frequency of single nucleotide mutation A/G at IVS8-281 is significantly different from that of the multi-lumbar phenotype (p < 0.01), indicating that it may affect the number of lumbar vertebrae. The follow-up study will further explore the significance of polymorphism sites for sheep breeding, and provide genetic markers for breeding new varieties to assist the breeding process of sheep.

Consent to participate
All authors reviewed and approved the manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The data that support the conclusions of this study are given in the text and supplementary material of this article.