The objective of the current study was to analyze expression levels of synapse differentiation inducing 1-like
(SYNDIG1L) and unc-13 homolog C (UNC13C) genes in different tissues, while single-nucleotide polymorphisms
(SNPs) of two genes were associated with multiple thoracic vertebrae traits
in both Small-tailed Han sheep (STH) and Sunite sheep (SNT). The expression
levels of SYNDIG1L and UNC13C were analyzed in the brain, cerebellum, heart, liver, spleen,
lung, kidney, adrenal gland, uterine horn, longissimus muscle, and abdominal
adipose tissues of two sheep breeds with different thoracic vertebral
number (TVN) sheep (T13 groups and T14 groups) by real-time quantitative
polymerase chain reaction (RT-qPCR). Meanwhile, the polymorphisms of UNC13C gene g.52919279C>T
and SYNDIG1L gene g.82573325C>A in T14 and T13 were
genotyped by the Sequenom MassARRAY® SNP assay, and
association analysis was performed with the TVN. The results demonstrated
that UNC13C gene was extensively expressed in 11 tissues. The expression of
UNC13C gene in longissimus muscle of T14 groups of STH was significantly higher
than that of T13 groups (P<0.05). SYNDIG1L gene was overexpressed in brain
and cerebellum tissues, and the expression level of UNC13C gene in the brain and
cerebellum of T13 groups in SNT was significantly higher than that of T14
groups (P<0.01). Association analysis showed that SNPs found in the
UNC13C gene had no significant effects on TVN for both two genes. The polymorphism
of SYNDIG1L g.82573325C>A was significantly correlated with the TVN in
both STH (P<0.05) and SNT (P<0.01). Taken together, the
SYNDIG1L gene was related to thoracic vertebral development, and this variation may
be potentially used as a molecular marker to select the multiple thoracic
vertebrae in sheep.
Introduction
The spine of a vertebrate consists of a series of repeated vertebrae. Based
on morphological differences, the vertebrae were subdivided into five
distinct functional spinal regions: cervical, thoracic, lumbar, sacral, and
caudal (Donaldson et al., 2013). The number of vertebrae is relatively
conserved among mammalian species. However, the quantitative variations of
vertebrae (Sun et al., 2019) have been observed in pigs (Rohrer and
Nonneman, 2017), deer (Mizer and Wahl, 2018), humans (Ibrahim et al., 2013),
and sheep (Donaldson et al., 2013). In general, the vertebrae of sheep were
arranged from the neck to the sacral part according to 7 cervical vertebrae
(C), 13 thoracic vertebrae (T), 6 lumbar vertebrae (L), and 4 sacral
vertebrae (S), with a total of 30 vertebrae. Among them, mutations in the
thoracolumbar position were the most common (T14L6 or T13L7) (Zhang, 1996).
Multi-vertebrae sheep have advantages in adaptability and meat production
performance (Zhang et al., 1996). The cultivation of multi-spine sheep has
comprehensive benefits for the economy, society, and ecology. This is of great
significance to improve the quality and efficiency of the animal husbandry
industry.
Among domestic animals, the most extensive studies of vertebral number
variation have focused on pigs. Previous studies have reported quantitative
trait loci (QTL) for vertebral numbers in pigs by genome scans based on
microsatellite markers. Two genome-wide significant QTLs were detected on
pig chromosomes (SSCs) 1 and 2 in a Meishan and Göttingen cross line
(Wada et al., 2000). The vertebrae-development-associated (VRTN) gene on the SSC7 and NR6A1 gene on SSC1 were
considered as candidate genes affecting vertebral numbers. Fine mapping of
vertebral number trait was performed, and an orphan nuclear receptor, germ
cell nuclear factor (NR6A1) was localized to be the candidate and also
confirmed by multiple studies (Mikawa et al., 2011; Zhang et al., 2015),
which was confirmed in various studies (Fan et al., 2013; Rohrer et al.,
2015; Yang et al., 2016). However, current studies on sheep vertebral number
traits were superficial, and functional studies focused on genomic
variations were relatively rare (Cao et al., 2015; Chen et al., 2012).
Small-tailed Han sheep (STH) selected in this study is one of the famous indigenous sheep breeds of
China which grows fast and has good early puberty and high fecundity (Guo et al.,
2020). Sunite sheep (SNT) are also an indigenous breed that has the advantages of
cold/drought resistance, fast growth, and good disease resistance. Meat
production performance is good, lean meat percentage is high, protein
content is high, it has a high popularity, and it has a large demand space at
home and even abroad (Zhong et al., 2020; Gao et al., 2014).
There was a high proportion of multiple thoracic and lumbar vertebral
numbers in both sheep breeds. Identification of molecular markers of
multi-spine variation for marker-assisted selection is of great significance
for the improvement of meat production performance.
Previously, we conducted a genome-wide association analysis in two sheep
breeds of 670 sheep with different thoracic vertebra numbers using a
Affymetrix ovine 600K single-nucleotide polymorphism
(SNP) array. Genome-wide significant associations were
detected at nine SNPs in the 245 kb region with p<1.13×10-9 (Yingjie Zhong, unpublished
data).
The significant SNPs on chromosome 7 were located near the region of
synapse differentiation inducing 1-like (SYNDIG1L) and unc-13 homolog C (UNC13C).
Whole-genome resequencing was also performed on 40 sheep with thoracic
vertebra numbers for fine mapping. Using the top 10 % of Fst values as
cutoffs, candidate genes associated with thoracic vertebra number were
identified, which included SYNDIG1L and UNC13C genes. Annotation of the sheep reference
genome (Oar4.0) suggested that the two non-synonymous mutations are located
in protein-coding regions of synapse differentiation inducing 1-like
SYNDIG1L and UNC13C genes respectively. g.82573325C>A located on exon 3 of
SYNDIG1L is a non-synonymous mutation that changes amino acid position 186 from
glycine (G) to tryptophan (W), while UNC13C g.52919279C>T located on
exon 14 is also a non-synonymous mutation that changes amino acid position
1465 from valine (V) to isoleucine (I).
The purpose of this study is to explore the association of UNC13C g.52919279C>T
and SYNDIG1L g.82573325C>A loci with thoracic vertebral
number. It provides promising candidate causal mutations for further
research on the number of vertebral variations on sheep.
Materials and methodsAnimal and main reagent
All the experimental procedures mentioned in the present study were approved
by the Science Research Department (in charge of animal welfare issues) of
the Institute of Animal Science, Chinese Academy of Agricultural Sciences
(IAS-CAAS) (Beijing, China). In addition, there was ethics
approval by the animal ethics committee of IAS-CAAS (no. IAS2020-82, 28 July 2020).
A total of 12 healthy ewes aged 3 years old were selected from the livestock and
breeding base of Tianjin Animal Husbandry and Veterinary Research Institute.
The number of thoracic vertebrae of SNT and STH was 13 and 14, respectively.
After slaughter, 11 tissues of brain, cerebellum, heart, liver, spleen,
lung, kidney, adrenal gland, uterine horn, longissimus muscle, and abdominal
fat were quickly collected, put into a 2 mL RNase-free centrifuge tube, and
stored in liquid nitrogen immediately. After returning to the laboratory,
they were stored in the freezer at -80∘C.
For genotyping (Table 1), a total of 383 sheep were selected from SNT and STH
from Bayan Nur slaughterhouses in the Inner Mongolia autonomous region, China,
and Yuncheng slaughterhouses in Shandong province. After slaughter, the
collected fresh muscle tissue was quickly put into a 2 mL frozen storage
tube and immediately stored in liquid nitrogen. After being brought back to
the laboratory, all the fresh muscle tissue was transferred to a freezer at
-80∘C for storage.
Sample information of the real-time quantitative
polymerase chain reaction (RT-qPCR) and genotyping.
BreedThoracicTissueRT-qPCRGenotypingvertebral no.sample no.sample no.SNT13Brain, cerebellum, heart, liver,312214spleen, lung, kidney, adrenal,366STH13uterine horn, longissimus muscle,313714abdominal fat358Total12383Extraction of genomic DNA and total RNA and main reagents
DNA from muscle tissue was extracted by a DNA extraction kit (TIANGEN
Biotech, Beijing, China). The total RNA of tissue was extracted using the Trizol
and Qiagen RNeasy kit (Qiagen). The concentration and integrity of DNA and
RNA were detected by Nanodrop2000, and the quality of DNA and RNA was
detected by 1.5 % agarose gel electrophoresis.
Quantitative polymerase chain reaction (PCR) was done using the SYBR Green fluorescent dye for product
detection (SYBR® Premix Ex Taq™ II). The
PrimeScript™ RT reagent kit was used to synthesize cDNA (TaKaRa,
Beijing).
cDNA synthesis
The total volume of the reaction system was 20 µL / 4.0 µL
5× PrimeScript buffer (for real time), 1.0 µL PrimeScript RT
enzyme mix E, 1.0 µL Oligo dT primer, 1.0 µL random 6 mers,
1000 ng RNA. The remaining system was supplemented with RNase-free
ddH2O. The reaction condition of PCR was 37 ∘C for 15 min and
85 ∘C for 5 s. The product obtained after reverse transcription
was diluted five times and stored in a freezer at -20∘C for detection
of tissue expression of the target gene.
Primer design
Primers for real-time quantitative
polymerase chain reaction (RT-qPCR) were designed using the GenBank database
(https://www.ncbi.nlm.nih.gov/genbank, last access: 10 July 2020). Genes and their accession numbers
include SYNDIG1L (GenBank: XM_027972017.1), UNC13C (GenBank: XM_027971817.1).
The β-actin (GenBank: NM_001009784.2) was an
internal reference gene. The primers (Table 2) were synthesized by Beijing
Tianyi Huiyuan Biotechnology Co., Ltd.
Using a Roche Light Cycler® 480 type II
fluorescence quantitative PCR instrument, the whole PCR process was
monitored in real time by fluorescence signal accumulation, and β-actin was used as an internal reference gene. The total volume of the reaction
system was 20 µL: SYBR Premix Ex Taq II 10 µL,
forward primer 0.8 µL, reverse primer 0.8 µL, RNase-free
ddH2O 6.4 µL, and cDNA 2.0 µL. PCR conditions were as
follows: initial denaturation at 95 ∘C for 5 s, followed by 40 cycles of 95 ∘C for 10 s and 60 ∘C for 30 s.
Genotyping
Genotyping of UNC13C g.52919279C>T and SYNDIG1L g.82573325C>A
was carried out using the Sequenom MassARRAY® SNP
(Johansen et al., 2013; Ortega et al., 2017) assay. The primer information is
provided in Table 3. The typing sample is DNA, and the amount required for
each sample is 20 µL. DNA concentration ranged from 40 to 80 ng/ µL.
The relative expressions of UNC13C and SYNDIG1L were calculated by the 2-ΔΔCt
method. The difference of relative expression between the T13 group and the
T14 group was analyzed by one-way ANOVA. The allele frequency, genotype
frequency, p value, polymorphism information content (PIC), heterozygosity
(He), and effective allele number (Ne) were calculated using Microsoft Excel
2016 statistical software. Then, the distribution of genotypes for each SNP
in the studied populations was tested for deviation from Hardy–Weinberg
equilibrium. P>0.05 indicates the locus was under Hardy–Weinberg
equilibrium.
The correlation between SNP and thoracic vertebra number traits of two
varieties was analyzed by SAS (V.9.4) (SAS Institute Inc.). The p values < 0.05 were considered to be significant. The mathematical models
are as follows: Fisher's exact probability test and logistic regression.
Logistic regression model:Logit(y)=lny1-y=β0+β1X1+β2X2+…+βnXn+e
Here, y represents the number of thoracic vertebrae, X1 represents the
variety, and X2 represents the genotype.
ResultsPolymorphism analysis of SYNDIG1L and UNC13C genes
The genotyping results of 383 sheep (Fig. 1) showed that two candidate loci
were polymorphic (Table 4). UNC13C g.52919279C>T and SYNDIG1L g.82573325C>A
displayed low polymorphisms (PIC< 0.25) in both SNT and
STH populations. Statistical significance was analyzed by the chi-square
test. The frequency of SYNDIG1L g.82573325C>A was consistent with
Hardy–Weinberg equilibrium in SNT, ensuring the reliability of their
application to evaluating larger groups (P>0.05). SYNDIG1L g.82573325C>A was, however, under Hardy–Weinberg imbalance (P<0.05) in the STH population. UNC13C g.52919279C>T satisfied
the Hardy–Weinberg equilibrium in both populations (P>0.05).
Genotyping results. (a) Scatter plot of SYNDIG1L genotyping results. (b) Scatter plot of UNC3C genotyping results.
Population genetic analysis of candidate loci in two sheep breeds.
GeneSNPBreedSample TotalGenotype Gene PICHeNepsize frequency frequency TTCTCCTTCTCCTCUNC13Cg.52919279C>TSNT4381431850.020.210.770.120.880.190.221.280.441STH3431491950.020.220.760.130.870.200.221.280.959AACACCAACACCACSYNDIG1Lg.82573325C>ASNT1321521850.010.170.820.090.910.150.171.200.620STH4231681950.020.120.860.080.920.140.151.170.007
He: heterozygosity; PIC: polymorphic information content; Ne: number of effective
alleles; P>0.05 indicates the locus was under Hardy–Weinberg equilibrium.
Association analysis of SYNDIG1Land UNC13C genes with thoracic vertebral number in two
breeds
Firstly, association analysis of SNPs with thoracic vertebral
number (TVN) was explored in two sheep
breeds, respectively. The statistical results were shown in Table 5.
UNC13C g.52919279C>T had no significant effect on TVN in both the STH
and SNT populations (P>0.05). SYNDIG1L g.82573325C>A was
significantly correlated with different thoracic vertebral numbers in both
STH (P<0.05) and SNT (P<0.01). Then, logistic regression
was used to test the effects of breeds and genotypes on different thoracic
vertebral numbers in sheep, which was shown in Table 6. For the two candidate
SNPs, the breeds have no significant relevance with TVN in sheep (P>0.05). The genotypes of SYNDIG1L g.82573325C>A were
significantly associated with multiple thoracic vertebrae in sheep (P<0.01), indicating that this gene might be correlated with a TVN
trait in the sheep.
Genotypes of candidate locus and the number of thoracic vertebrae in
a single breed by Fisher's exact test.
P≤0.05 indicates the significant difference; P≤0.01 indicates the
extremely significant difference.
Expression profiles of UNC13C and SYNDIG1L genes in SNT and STH with different TVNs
The results of RT-qPCR showed that the UNC13C gene was extensively expressed in 11
tissues of STH and SNT (Fig. 2a). The SYNDIG1L gene was mainly expressed in brain
tissue and slightly expressed in the spleen in SNT with T13 (Fig. 2b).
The UNC13C gene was highly expressed in the cerebellum of STH. The expression of
UNC13C in group T14STH was significantly higher than group T13STH in the
longissimus muscle (P<0.05); the gene expression fold change is
1.8. In T13SNT, the expression of the SYNDIG1L gene in the brain and cerebellum tissues
was significantly higher than that in T14SNT (P<0.05); the fold
changes of gene expression were 0.5 and 0.4, respectively.
Results of expression level of UNC13C and SYNDIG1L genes in STH and SNT with different TVNs.
(a) The comparison of expression levels of UNC3C between SNT and STH. (b) The
comparison of expression levels of SYNDIG1L between SNT and STH. The significant
results with a p value lower than 0.05 are given one asterisk (∗).
Discussion
The vertebrae of mammals are derived from the mesoderm of the gastrula.
Vertebrae development is an extremely complicated system that is regulated
temporally and spatially. It has been known that any error in development
can result in many congenital abnormalities (Gilbert, 2003). Zhang et al. (1998) found that the meat-production performance of
multi-vertebrae sheep was significantly better than that of normal sheep, with
longer longissimus muscle, larger abdominal cavity volume, carcass weight,
net meat weight, lean meat percentage, and other economic indexes. Moreover,
this trait is heritable. Thus, it is important to understand the mechanism
of vertebral number variation from the molecular level and apply it to sheep
breeding with multiple thoracic vertebrae. The genetic architecture of
thoracic vertebral number has been extensively studied in pigs, and major
genes affecting this trait have been mapped in indigenous pigs (Rohrer et
al., 2015; Duan et al., 2018; Liu et al., 2020). However, current studies on
sheep vertebral numbers were superficial, and functional studies focused on
genomic variations were relatively rare.
Liu et al. (2020) found that regulation variants on SSC7 might
play crucial roles in the number of thoracic vertebrae (NTV)
and the FOS (Fos proto-oncogene, AP-1 transcription factor subunit) on SSC7, and BMPR1A was identified as a
novel candidate gene affecting the NTV in pigs on SSC14. Fan et al. (2013)
identified three loci for a vertebral number trait through a genome-wide
association study and located them in a 947 kb region on SSC7 in pigs. The
locus was refined to 100 kb by a homologous sharing test, which contained only
VRTN and SYNDIG1L genes. Among them, VRTN is considered to be the main candidate gene
affecting vertebral numbers in the modern western world. The VRTN gene was considered
as a candidate gene affecting vertebral number also in sheep (Li et al.,
2019). We believe that it is not accidental that SYNDIG1L and VRTN have been identified
at the same time. SYNDIG1L is highly expressed in the striatum (de Chaldée et
al., 2006). This is consistent with our previous RT-qPCR results. As one of
the basal ganglia of the brain, the striatum is mainly responsible for
regulating muscle tension and coordinating various fine and complicated
movements (Lorenc-Koci et al., 1998; Hemsley and Crocker, 2001). Meanwhile,
it is related to the occurrence of Parkinson's disease (Miyanishi et al.,
2019; Choe et al., 2011), chorea (Ishikawa et al., 1990), and other
diseases. Correspondingly, some researchers found that the incidence of
dyskinesia increased with the increase of thoracic vertebral numbers in pigs
(Nakano et al., 2015). This may be due to the negative effects of high
expression of SYNDIG1L. On the other hand, SYNDIG1L was reported to be a factor affecting
the final body weight and back-fat thickness in Landrace pigs (Lee et al.,
2018). An et al. (2020) believe that it is the key gene that
affects the formation of bovine body shape. We speculate that SYNDIG1L may
participate in the spine formation process and cause mutation in SYNDIG1L, which may lead
to abnormal development of vertebrae in sheep. However, more evidence is needed
to prove our hypothesis.
The new role of the UNC13C gene in oral squamous cell carcinoma (OSCC) has been
revealed for the first time. UNC13C is a novel tumor suppressor and can be used as
a target to prevent oral cancer metastasis (Velmurugan et al., 2019). Studies
have shown that UNC13C is involved in Alzheimer's disease (AD), which involves
dysfunction of many cellular pathways, including synaptic transmission,
cytoskeleton dynamics, energetics, and apoptosis (Miller et al., 2013).
According to references, UNC13C is significantly downregulated in spinal cord
tissue of patients with amyotrophic lateral sclerosis (D'Erchia et al.,
2017). It is considered to be negatively correlated with muscle ability in
the study of myasthenia (Hangelbroek et al., 2016), no direct relationship
between the function of the UNC13C gene and the number of thoracic vertebrae was
found.
Conclusion
This study found that the polymorphisms of SYNDIG1L g.82573325C>A were
significantly associated with the thoracic vertebral number in sheep,
indicating that this locus may be a promising candidate causal variation in
the regulation of thoracic vertebral numbers. Further exploration of the
functions of the SYNDIG1L gene was necessary for the cultivation of sheep breeds with
multiple thoracic vertebrae.
Data availability
The data sets are available upon request from the corresponding authors.
Author contributions
YJZ and QYL contributed to the conception of the study.
YY contributed significantly to analysis and manuscript preparation.
YJZ performed the data analyses and wrote the manuscript.
MXC contributed to revisions of the manuscript. XYW and RD assisted the analysis with constructive discussion.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
We thank Mingxing Chu, Qiuyue Liu, and all the faculties involved including Chinese Academy of Agricultural Sciences, as well as the local abattoir for their support during this study.
Financial support
This work was supported by the Genetically Modified Organisms Breeding Major
Program of China (grant no. 2016ZX08009-003-006), the Central Public-interest
Scientific Institution Basal Research Fund (grant no. 2017ywf-zd-13), the Agricultural
Science and Technology Innovation Program of China (grant no. ASTIP-IAS13), and the
Earmarked Fund for China Agriculture Research System (grant no. CARS-38).
Review statement
This paper was edited by Steffen Maak and reviewed by two anonymous referees.
ReferencesAn, B., Xu, L., Xia, J., Wang, X., Miao, J., Chang, T., Song, M., Ni, J.,
Xu, L., Zhang, L., Li, J., and Gao, H.: Multiple association analysis of
loci and candidate genes that regulate body size at three growth stages in
Simmental beef cattle, BMC Genet., 21, 32, 10.1186/s12863-020-0837-6, 2020.Cao, J., Wei, C., Liu, D., Wang, H., Wu, M., Xie, Z., Capellini, T. D.,
Zhang, L., Zhao, F., Li, L., Zhong, T., Wang, L., Lu, J., Liu, R., Zhang,
S., Du, Y., Zhang, H., and Du, L.: DNA methylation landscape of body size
variation in sheep, Sci. Rep.-UK, 5, 13950, 10.1038/srep13950,
2015.Chen, Q., Zhang, L. L., Zhao, J., and Ma, Y. H.: DNA methylation analysis of exon-1 of the ovine HOXC-8 gene in
Mongolian sheep using bisulphite sequencing, J. Appl. Anim. Res., 40, 198–202,
10.1080/09712119.2012.658059, 2012.Choe, M. A., An, G. J., Koo, B. S., and Jeon, S.: Effect of DHEA on recovery
of muscle atrophy induced by Parkinson's disease, J. Korean Acad. Nurs., 41,
834–842, 10.4040/jkan.2011.41.6.834, 2011.de Chaldée, M., Brochier, C., Van de Vel, A., Caudy, N., Luthi-Carter,
R., Gaillard, M. C., and Elalouf, J. M.: Capucin: a novel striatal marker
down-regulated in rodent models of Huntington disease, Genomics, 87,
200–207, 10.1016/j.ygeno.2005.10.009, 2006.D'Erchia, A. M., Gallo, A., Manzari, C., Raho, S., Horner, D. S., Chiara,
M., Valletti, A., Aiello, I., Mastropasqua, F., Ciaccia, L., Locatelli, F.,
Pisani, F., Nicchia, G. P., Svelto, M., Pesole, G., and Picardi, E.: Massive
transcriptome sequencing of human spinal cord tissues provides new insights
into motor neuron degeneration in ALS, Sci. Rep.-UK, 7, 10046,
10.1038/s41598-017-10488-7, 2017.Donaldson, C. L., Lambe, N. R., Maltin, C. A., Knott, S., and Bunger, L.:
Between- and within-breed variations of spine characteristics in sheep, J.
Anim. Sci., 91, 995–1004, 10.2527/jas.2012-5456, 2013.Duan, Y., Zhang, H., Zhang, Z., Gao, J., Yang, J., Wu, Z., Fan, Y., Xing,
Y., Li, L., Xiao, S., Hou, Y., Ren, J., and Huang, L.: VRTN is required for
the development of thoracic vertebrae in mammals, Int. J. Biol. Sci., 14,
667–681, 10.7150/ijbs.23815, 2018.Fan, Y., Xing, Y., Zhang, Z., Ai, H., Ouyang, Z., Ouyang, J., Yang, M., Li,
P., Chen, Y., Gao, J., Li, L., Huang, L., and Ren, J.: A further look at
porcine chromosome 7 reveals VRTN variants associated with vertebral number
in Chinese and Western pigs, PLoS ONE, 8, e62534,
10.1371/journal.pone.0062534, 2013.
Gao, F. M., Bai, Y. E. T., Liu, J., Sun, Y. J., and Shao, Z. Y.: Quality and
nutrition of Sunite sheep and mutton, Abstract of Animal Husbandry and
Veterinary of China, 30, 44–43,
https://doi.org/CNKI:SUN:ZXWA.0.2014-12-040, 2014.
Gilbert, S. F.: The morphogenesis of evolutionary developmental biology, Int.
J. Dev. Biol., 47, 467–477, 2003.
Guo, D. E., Wang, W. K., Cui, B. H., Li, J., and Tang, l.: Comparison of
production performance between Small tail Han sheep and Hu sheep, Jilin
Animal Husbandry and Veterinary Medicine, 41, 60, 2020.Hangelbroek, R. W., Fazelzadeh, P., Tieland, M., Boekschoten, M. V.,
Hooiveld, G. J., van Duynhoven, J. P., Timmons, J. A., Verdijk, L. B., de
Groot, L. C., van Loon, L. J., and Müller, M.: Expression of
protocadherin gamma in skeletal muscle tissue is associated with age and
muscle weakness, J Cachexia Sarcopenia Muscle, 7, 604–614,
10.1002/jcsm.12099, 2016.Hemsley, K. M. and Crocker, A. D.: Changes in muscle tone are regulated by
D1 and D2 dopamine receptors in the ventral striatum and D1 receptors in the
substantia nigra, Neuropsychopharmacology: Neuropsychopharmacology, 25,
514–526, 10.1016/s0893-133x(01)00245-7, 2001.Ibrahim, D. A., Myung, K. S., and Skaggs, D. L.: Ten percent of patients
with adolescent idiopathic scoliosis have variations in the number of
thoracic or lumbar vertebrae, J. Bone Joint Surg. Am., 95, 828–833,
10.2106/jbjs.L.00461, 2013.
Ishikawa, A., Miyatani, N., Yuasa, T., Tanaka, K., and Oyanagi, K.: An
autopsied case of manifesting chorea, serum antibody to brain proteins,
neuronal degeneration in striatum and grumose degeneration in dentate
nucleus, Rinsho Shinkeigaku, 30, 510–515, 1990.Johansen, P., Andersen, J. D., Børsting, C., and Morling, N.: Evaluation
of the iPLEX® Sample ID Plus Panel designed for the Sequenom
MassARRAY® system. A SNP typing assay developed for human
identification and sample tracking based on the SNPforID panel, Forensic
science international, Forensic. Sci. Int. Genet., 7, 482–487,
10.1016/j.fsigen.2013.04.009, 2013.Lee, Y. S., Shin, D., and Song, K. D.: Dominance effects of ion transport
and ion transport regulator genes on the final weight and backfat thickness
of Landrace pigs by dominance deviation analysis, Genes Genomics, 40,
1331–1338, 10.1007/s13258-018-0728-7, 2018.Li, C. Y., Li, M., Li, X. Y., Ni, W., Xu, Y. R., Yao, R., Wei, B., Zhang,
M. D., Li, H. X., Zhao, Y., Liu, L., Yaseen, U., Jiang, Y., and Hu, S. W.: Whole-genome
resequencing reveals loci associated with thoracic vertebrae number in
sheep, Front. Genet., 18, 674, 10.3389/fgene.2019.00674, 2019.Liu, Q., Yue, J. W., Niu, N. Q., Liu, X., Yan, H., Zhao, F. P., Hou, X. H., Gao, H.
M., Shi, L. J., Wang, L. X., Wang, L. G., and Zhang, L. C.: Genome-wide
association analysis identified BMPR1A as a novel candidate gene affecting the
number of thoracic vertebrae in a Large White × Minzhu intercross
pig population. Animals (Basel), 10, 2186,
10.3390/ani10112186, 2020.Lorenc-Koci, E., Konieczny, J., and Wolfarth, S.: Contribution of the
glycine site of NMDA receptors in rostral and intermediate-caudal parts of
the striatum to the regulation of muscle tone in rats, Brain Res., 793,
315–320, 10.1016/s0006-8993(98)00240-6, 1998.Mikawa, S., Sato, S., Nii, M., Morozumi, T., Yoshioka, G., Imaeda, N.,
Yamaguchi, T., Hayashi, T., and Awata, T.: Identification of a second gene
associated with variation in vertebral number in domestic pigs, BMC Genet.,
12, 5, 10.1186/1471-2156-12-5, 2011.Miller, J. A., Woltjer, R. L., Goodenbour, J. M., Horvath, S., and
Geschwind, D. H.: Genes and pathways underlying regional and cell type
changes in Alzheimer's disease, Genome Med., 5, 48,
10.1186/gm452, 2013.Miyanishi, K., Choudhury, M. E., Watanabe, M., Kubo, M., Nomoto, M., Yano,
H., and Tanaka, J.: Behavioral tests predicting striatal dopamine level in a
rat hemi-Parkinson's disease model, Neurochem Int., 122, 38–46,
10.1016/j.neuint.2018.11.005, 2019.Mizer, L. A. and Wahl, C.: The noncervical lateral transverse foramina, J
Morphol, 279, 1679–1691, 10.1002/jmor.20905, 2018.Nakano, H., Sato, S., Uemoto, Y., Kikuchi, T., Shibata, T., Kadowaki, H.,
Kobayashi, E., and Suzuki, K.: Effect of VRTN gene polymorphisms on Duroc
pig production and carcass traits, and their genetic relationships, Anim. Sci.
J., 86, 125–131, 10.1111/asj.12260, 2015.Ortega, M. S., Denicol, A. C., Cole, J. B., Null, D. J., Taylor, J. F.,
Schnabel, R. D., and Hansen, P. J.: Association of single nucleotide
polymorphisms in candidate genes previously related to genetic variation in
fertility with phenotypic measurements of reproductive function in Holstein
cows, J. Dairy Sci., 100, 3725–3734, 10.3168/jds.2016-12260,
2017.Rohrer, G. A. and Nonneman, D. J.: Genetic analysis of teat number in pigs
reveals some developmental pathways independent of vertebra number and
several loci which only affect a specific side, Genet. Sel. Evol., 49, 4,
10.1186/s12711-016-0282-1, 2017.Rohrer, G. A., Nonneman, D. J., Wiedmann, R. T., and Schneider, J. F.: A
study of vertebra number in pigs confirms the association of vertnin and
reveals additional QTL, BMC Genet., 16, 129,
10.1186/s12863-015-0286-9, 2015.Sun, Q., Liu, S. J., Di, R., Hu, W. P., Wang, X. Y., Ma, L., Zhang, X. S.,
Zhang, J. L., Liu, Q. Y., and Chu, M. X.: Association between polymorphism
of VRTN, NR6A1 genes and thoracic vertebral number variation and analysis of
their tissue expression in Sunite sheep (Ovis aries), Journal of Agricultural
Biotechnology, 27, 864–874, https://doi.org/CNKI:SUN:NYSB.0.2019-05-010,
2019.Velmurugan, B. K., Yeh, K. T., Hsieh, M. J., Yeh, C. M., Lin, C. C., Kao, C.
Y., Huang, L. R., and Lin, S. H.: UNC13C suppress tumor progression via
inhibiting EMT pathway and improves survival in oral squamous cell
carcinoma, Front Oncol., 9, 728, 10.3389/fonc.2019.00728,
2019.Wada, Y., Akita, T., Awata, T., Furukawa, T., Sugai, N., Inage, Y., Ishii,
K., Ito, Y., Kobayashi, E., Kusumoto, H., Matsumoto, T., Mikawa, S., Miyake,
M., Murase, A., Shimanuki, S., Sugiyama, T., Uchida, Y., Yanai, S., and
Yasue, H.: Quantitative trait loci (QTL) analysis in a Meishan x
Göttingen cross population, Anim. Genet., 31, 376–384,
10.1046/j.1365-2052.2000.00696.x, 2000.Yang, J., Huang, L., Yang, M., Fan, Y., Li, L., Fang, S., Deng, W., Cui, L.,
Zhang, Z., Ai, H., Wu, Z., Gao, J., and Ren, J.: Possible introgression of
the VRTN mutation increasing vertebral number, carcass length and teat
number from Chinese pigs into European pigs, Sci. Rep., 6, 19240,
10.1038/srep19240, 2016.Zhang L. C., Liu X., Liang J., Yan H., Zhao K. B., LI N., PU L., Shi H. B., Zhang Y. B., Wang L. G.: Quantitative
trait loci for the number of vertebrae on Sus scrofa chromosomes 1 and 7
independently influence the numbers of thoracic and lumbar vertebrae in
pigs, J. Integr. Agr., 14, 2027–2033,
10.1016/S2095-3119(15)61084-X, 2015.
Zhang, L. L.: Genetic mechanism of polyspinous phenomena in Mongolian sheep,
China Sheep, 6, 1–3, 1996.
Zhang, L. L., Ji, E. G. L., and Zhou, W.: Study on population Genetic
characteristics of spinal number variation in Wuzhu Muqin sheep, Animal
Husbandry and Feed Science, 000, 1–3, 1996.
Zhang, L. L., Luo, X. G., Si, Q. B. L. G., and Zhang, S. S.: Analysis of
thoracolumar and lumbar spine length and meat production performance of
Mongolian sheep with multiple vertebrae, Journal of Inner Mongolia
Agricultural University (Natural Science Edition), 19, 1–5, 1998.Zhong, Y. J., Xiang, G. M., He, X. Y., Liu, S. J., Zhang, X. S., Zhang, J.
L., Chu, M. X., and Liu, Q. Y.: Preliminary study on the relationship
between FBXL3 and FBXL21 gene expression and seasonal estrus in Sunite
sheep, Chinese Journal of Animal Science, 56, 1–10,
10.19556/j.0258-7033.20200107-01, 2020.