AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-61-1-2018A novel 12 bp deletion within goat LHX4 gene significantly affected litter sizeYanHailongZhangFeiWangKeLiuJinwangZhuHaijingPanChuanyingQuLeiylqulei@126.comShaanxi Provincial Engineering and Technology Research Center of
Cashmere Goats, Yulin University, Yulin 719000, ChinaLife Science Research Center, Yulin University, Yulin 719000, ChinaCollege of Animal Science and Technology, Northwest A&F University,
Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling,
712100 Shaanxi, ChinaInnovation Experimental College, Northwest A&F University, Yangling
712100, ChinaThese authors contributed equally to this work.Lei Qu (ylqulei@126.com)4January20186111828August20173November201714November2017This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/This article is available from https://aab.copernicus.org/articles/61/1/2018/aab-61-1-2018.htmlThe full text article is available as a PDF file from https://aab.copernicus.org/articles/61/1/2018/aab-61-1-2018.pdf
The LIM homeobox transcription factor 4 (LHX4) gene plays a critical role in
regulating the development of the pituitary and the secretion of growth hormone (GH) and prolactin
(PRL) associated with reproduction. Thus this gene may affect litter size. Herein,
the aim of this study is to detect the novel insertion/deletion (indel)
within the LHX4 gene as well as to test its association with litter
size in 1149 Shaanbei white cashmere goats. Herein, a novel 12 bp indel
(NC_030823.1:g.60001011_60001022delGGGGAGGAGGGG) was firstly found,
which was located in the first intron. Meanwhile, three genotypes were detected
in Shaanbei white cashmere goats, and the allelic frequencies of I and
D were 0.593 and 0.407, respectively. Interestingly, the genotype
distributions between mothers of single-lamb (n=895) and multi-lamb (n=254) groups within Shaanbei white cashmere goats were significantly
different, implying that the 12 bp indel might affect the litter size.
Furthermore, the association analysis was carried out to find out that the
12 bp indel was significantly associated with litter size in the analyzed
goat population (P< 0.05). The litter sizes of genotype DD and ID
individuals were superior to those of genotype II (P< 0.05). These
findings suggest that this locus could be considered as a genetic marker
for goat breeding, enriching the research category of functional genome of
goats.
Introduction
Along with the rapid development of “The Belt and Road” policy and
improvement of people's living standards, the demand for goat products is
increasing in numerous developing countries, especially in China. However,
China is experiencing a severe shortage of goat products. Litter size, as one
of the most important reproductive and economic traits, is a very critical
factor for increasing goat industry. However, it is difficult to improve
litter size rapidly using traditional methods because the small size is
controlled by multiple genes. Thus using DNA selection via related genes is
becoming more and more necessary (Zhang et al., 2015). To date, many
important potential molecular markers for goat marker-assisted selection
(MAS), including single nucleotide polymorphism (SNP) and insertion/deletion
(indel), have been performed by previous studies. For example, the goat
INHA 651A/G polymorphism significantly affected the litter size in
Boer goats (Wu et al., 2009). The polymorphism in the promoter region of the
KISS1 gene has a notable correlation with the litter size (An et
al., 2015a). Additionally, FTH1, growth hormone (GH), and serum amyloid A (SAA) genes were significantly associated with high litter size in Jining Grey goats (Feng et al., 2015). Most studies concentrated on SNP related to litter
size trait (Li et al., 2008; An et al., 2013a), however, few on indel. For
the indel marker, there were several advantages for MAS breeding, such as
simple operation, rapid detection, and easy utilization (Jin et al., 2016;
M. Zhang et al., 2016). Hence, it is necessary to find the novel indel within
the candidate genes associated with litter size in goat industry in the
future.
As a member of the LIM-HD gene family, the LIM homeobox transcription factor
4 (LHX4) gene plays an important role in regulating the development
of the pituitary and nervous system, as well as in participating in the
LHX3–LHX4–PROP1–POU1F1 pathway (Wu et al., 1998; Sloop et al.,
2000). Notably, the pituitary is one of the most important endocrine glands
of the hypothalamic–pituitary–gonadal (HPG) axis and has a critical effect
on reproduction. Thus, it could be indicated that the LHX4 gene is
an excellent candidate gene for reproductive traits in mammals. Previous
studies have manifested that the LHX4 gene regulates the secretion
of hormones, such as follicle-stimulating hormone (FSH), GH, luteinizing
hormone (LH), thyroid-stimulating hormone (TSH), and prolactin (PRL) by
acting on the pituitary gland directly or indirectly. These hormones regulate
growth and metabolism, reproductive development, and so on in humans and
livestock (Mullena et al., 2007). Deficiency of the LHX4 and other
genes (such as LHX3, and Pitx2) renders combined pituitary
hormone deficiency (CPHD) and pituitary hypoplasia in both humans and mice
(Raetzman et al., 2002; Hunter and Rhodes, 2005; Pfaeffle et al., 2008),
suggesting that the LHX4 gene has a significant influence on
stimulating the rapid proliferation of undifferentiated pituitary progenitors
via activating LHX3 and maintaining expression of Pitx2 in
mice (Gergics et al., 2015). Moreover, the mutations of the LHX4
gene are also associated with dominantly inherited GH deficiency. To date,
LHX4-driven pathway could have influenced the expression of GH
(Machinis and Amselem, 2005), POU1F1, PRL, and other genes which are closely
related to reproduction of livestock (Wu and Xu, 2000; Lan et al., 2007; Yang
et al., 2017). Therefore, the LHX4 gene was possibly associated with
CPHD and reproduction traits in livestock.
To date, the polymorphisms of the bovine LHX4 gene have been found,
and they were associated with growth traits (Ren et al., 2014). A missense
mutation within the goat LHX4 gene was reported, but its function
was unknown (Li et al., 2008). Briefly, little information about the LHX4 gene indel variants and its association with reproduction traits was found. Therefore, in this work, the novel indel mutation of
the LHX4 gene in a Chinese indigenous goat breed was detected, and
its association with litter size was analyzed, which would benefit the
acquisition of potential useful DNA markers for goat MAS breeding, more so
than pushing “one belt and one road” in goat production.
Material and methods
All experimental animals in this study were approved by the Institutional
Animals Care and Use Committee (IACUC) of Northwest A&F University
(NWAFU). Furthermore, the use of experimental animals was in compliance with the
local animal welfare laws, guidelines, and policies.
DNA samples and related data collection
The ear tissue samples from a total of 1149 Shaanbei white cashmere goats
were obtained from a farm in central Yulin in Shaanxi Province (Wang et al.,
2017; Yang et al., 2017). All the goats were reared on the same farm under
normal conditions. Furthermore, the litter size of Shaanbei white cashmere
goats in the first birth was recorded.
Genotypic and allelic frequencies and other population indexes in
the Shaanbei white cashmere goat LHX4 gene.
SizeGenotypic frequency Allelic frequency P(HWE)HoHeNePICIIIDDDID11490.3450.4960.1590.5930.407P>0.050.5170.4831.9340.366
The genotype distribution between mothers of a single lamb and
multiple lambs in Shaanbei white cashmere goats.
TypesSampleGenotype Genotype Independentsizesnumbers frequencies χ2 value, df, P valueIIIDDDIIIDDDMothers of a single lamb8953294351310.3680.4860.146χ2=11.242Mothers of multiple lambs (≥ 2)25467135520.2640.5310.205df = 4P=0.036DNA extraction and genomic DNA pool construction
DNA samples were isolated from ear tissues using the approach of high salt
extraction and diluted to a specific concentration (10 ng µL)
(Yang et al., 2017). Fifty DNA samples were randomly selected and mixed into
the PCR tube, which could be used as templates to scan the indel mutation in
PCR amplification.
Primer design, PCR amplification, and DNA sequencing
According to the sequence of goat LHX4 gene (GenBank accession
number N_030823.1) in NCBI (www.ncbi.nlm.nih.gov), only one putative
indel sequence was provided. Hence, in this work, a pair of primers (F:
5′-AGCGAGGCAAGGCTGAAC-3′; R: 5′-GGGTCCTACATCCCAAGAAA-3′) were
designed using Primer Premier 5.0 software (Premier Biosoft International
USA) and synthesized by Sangon Biotech (Shanghai, China).
The PCR amplification was performed in a 12.5 µL of reaction volume
containing 1.5 µL genomic DNA (10 ng µL),
0.5 µL of each primer, 6 µL 2 ×Taq
Master Mix (BioLinker, Shanghai, China), 4 µL ddH2O. The
Touch-Down PCR (TD-PCR) program was performed as described previously:
initially denatured at 95 ∘C for 5 min; 2 cycles of 94 ∘C
for 30 s; annealing at 68 to 51 ∘C for 30 s (with a
decrease of 3 ∘C per 2 cycles); extended at 72 ∘C for
40 s; a final extension at 72 ∘C for 10 min and cooling to
10 ∘C (Xu et al., 2017). The amplification product was detected by
2.5 % agarose gel electrophoresis in 1 × TBE with constant
voltage (120 V) for about 55 min. At last, agarose gel was stained with
ethidium bromide and the PCR product was observed.
Statistical analysis
Genotypic and allelic frequencies of the goat LHX4 gene were
calculated directly. Hardy–Weinberg equilibrium (HWE) was performed by the SHEsis
program (http://analysis.bio-x.cn) (Li et al., 2009). Heterozygosity
(He), homozygosity (Ho), effective allele numbers (Ne), and polymorphism
information content (PIC) were calculated following Nei's method (Nei, 1973)
and performed on the program PopGene3.2. Distribution differences for
genotypic frequencies between the mothers of a single lamb and multiple lambs were
analyzed using the χ2 test, which was carried out using SPSS software
(version 21.0) (IBM Corporation, New York, USA). Meanwhile, a different
genotype was considered as an independent variable and litter size was used as
the dependent variable using the software program SPSS for correlation
analysis. The ANOVA applied to the general liner model (GML) was simplified as
follows: Yij=μ+Gi+Rj+Pk+e, where Yij is the
observation of the litter size, μ is the overall average number of
litter size, Gi is the fixed effect of genotype or combined genotype,
Rj is the effect of lambing year, Pk is the fixed effect of the
parity, and e stands for random residual error (He et al., 2014; Wang et al.,
2014; Yang et al., 2016).
The electrophoresis pattern with 2.5 % agarose gel of the 12 bp
indel within goat LHX4 gene. II = 303 bp; DD = 291 bp;
ID = 303 bp + 291 bp; M = DNA marker I.
The genotype distributions among mothers of a single lamb, two
lambs, and three lambs in Shaanbei white cashmere goats.
TypesSampleGenotype Genotype Independentsizesnumbers frequencies χ2 value, df, P valueIIIDDDIIIDDDMothers of a single lamb8953294351310.3680.4860.146χ2=15.803Mothers of two lambs23964129460.2680.5400.192df = 4Mothers of three lambs153660.2000.4000.400P=0.003
Sequencing graph of a 12 bp indel within goat LHX4 gene.
Panel (II): homozygous insertion genotype II, the sequence with a 12 bp
insertion. Panel (DD): homozygous deletion genotype DD.
ResultsIdentification of a 12 bp indel variation
Agarose gel electrophoresis and PCR production sequencing convinced a 12 bp
indel within the LHX4 gene (Figs. 1; 2). Furthermore, three
genotypes (II, ID, and DD) were identified. Genotype II exhibited one band of
303 bp, genotype DD exhibited one band of 291 bp, and heterozygote genotype
ID exhibited two bands (303 and 291 bp).
Genetic parameters analysis
Genotypic frequency, allele frequency, Ho, He, Ne, and PIC of tested goat
population were calculated and are shown in Table 1. The frequencies of II and ID
genotypes were higher than DD genotype in Shaanbei white cashmere goats.
I allele had a higher frequency than D. This indel locus was in
accord with Hardy–Weinberg equilibrium (HWE) in tested goat population (P>0.05). Moreover, the genotype distributions between mothers of
a single lamb (n=895) and multiple lambs (n=254) in Shaanbei white cashmere
goats were significantly different (P<0.05) (Tables 2; 3; Fig. 3).
The relationship between three genotypes of indel variation and
litter sizes in Shaanbei white cashmere goats (mean ± SE).
Note: cells with different letters
(a, b) mean P<0.05.
Relationship between a 12 bp indel and litter size
The correlation between 12 bp duplication indel of goats LHX4 gene
and litter size was conducted, and this 12 bp indel was revealed to show
remarkable association with litter size (P<0.05) (Table 4; Fig. 4).
Moreover, the individuals with the genotype DD have the highest average litter
size, followed by individuals with heterozygous ID genotype and lowest in II
genotype (P<0.05).
Discussions
The litter size traits are intricate quantitative traits involving multiple
genes and interactions (An et al., 2013a), so it is important to analyze the
inner connection of different genes. Previous studies detected that many
genetic mutations within candidate genes could affect goat litter size
traits. The genetic polymorphism of KISS1 gene was explored and
showed that four SNPs may affect litter size in goats (An et al., 2015a).
Polymorphisms of GNRH1 and GDF9 genes were identified, and
their association with litter size in goats was analyzed (An et al., 2013a).
The SNPs of the PRLR gene regulated by bta-miR-302a associated with
litter size in goats were analyzed (An et al., 2015b). The genetic
polymorphisms of KITLG gene were explored, and the results indicated
that three SNPs may play an important role in litter size (An et al., 2013b;
Wang et al., 2017). Otherwise, some candidate genes, such as FTH1, GH, and
SAA, were significantly associated with high litter size in Jining Grey goats
(Feng et al., 2015). The LHX4 gene, as a member of the LIM-HD gene
family, plays an important role in regulating the development of the
pituitary and nervous system and participating in
LHX3–LHX4–PROP1–POU1F1 pathway (Wu et al., 1998; Sloop et al.,
2000). On the one hand, LHX4 gene can stimulate the secretion of FSH
and LH by acting on the pituitary. FSH and LH have a direct effect on gonadal
development and then affect the litter size. On the other hand, LHX4
gene could participate in LHX3–LHX4–PROP1–POU1F1 pathway and then
have an influence on POU1F1. POU1F1 can affect the expression of GH, PRL, and ACTH (adrenocorticotropic hormone). GH directly affects
the growth and development of organisms. The previous research showed that
LHX4 gene had a notable association with growth traits in livestock
(Ren et al., 2014). Meanwhile, the POU1F1 can affect the embryonic
development and then have an influence on litter size. Therefore, this work
focused on detecting the potential indel variation within the LHX4
gene and its effects on litter size.
In this study, a novel 12 bp indel was verified. According to the
classification of PIC, it was found that this locus owned moderate genetic
diversity. Moreover, this indel was in Hardy–Weinberg equilibrium (HWE) (P>0.05), which shows the tested Shaanbei white cashmere goat
population was in a state of equilibrium. Through the χ2 test, the
significant genotypic and allelic distribution differences between mothers
of a single lamb (n=895) and multiple lambs (n=254) in Shaanbei white cashmere
goats were revealed, implying that this indel could affect litter size.
The percentage of allelotypes and alleles of mothers of a single
lamb and multiple lambs in Shaanbei white cashmere goats.
*P< 0.05, **P< 0.01.
Furthermore, using statistical analysis, this 12 bp indel within
LHX4 gene was significantly associated with litter size in goats.
The individuals with DD genotype had higher average litter size than those
with II and DD genotypes. Notably, D allele was the positive allele affecting
the litter size. A previous study showed litter sizes in sheep and goats were
regulated by the hypothalamic–pituitary–gonadal (HPG) axis, which
coordinates reproductive behavior with follicular development, ovulation,
fertilization, embryogenesis, and parturition (Feng et al., 2015). Although
the intron does not appear in the coding region, some conclusions indicated
that introns also acted as important gene regulatory elements. Van Laere et
al. (2003) discovered a paternally expressed quantitative trait locus (QTL),
which can affect muscle growth, fat deposition in pig maps in intron3 of
IGF2. Studies have shown that introns not only contain many gene
expression and regulation elements in relation to gene transcription and mRNA
processing especially the alternative splicing but also contain kinds of
non-coding RNA (Q. Zhang et al., 2016). Thus, this intronic indel within the
LHX4 gene might affect expression of the cell cycle regulators and
then have an influence on litter size in goats.
The association of the different genotypes and litter size in
Shaanbei white cashmere goats. *P< 0.05, **P< 0.01.
In summary, a 12 bp deletion of the LHX4 gene was significantly
associated with the litter size in goats, which would extend the indel
variations spectrum of the LHX4 gene and contribute to promising
indel markers in goat breeding.
The original data of the paper are available upon request
from the corresponding author.
The authors declare that they have no conflict of
interest.
Acknowledgements
This work was funded by the National Natural Science Foundation of China
(no. 31760650) and Provincial Key Projects of Shaanxi (2014KTDZ02-01). We
greatly thank the staff of a Shaanbei white cashmere goat breeding farm,
Shaanxi province, P.R. China, for collecting samples. Edited by: Steffen Maak Reviewed by: Zhuanjian
Li and one anonymous referee
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