Investigation Of MRNA Expression,InDel,and CNV In Six Candidate Genes And Their Associations With Litter Size In Shaanbei White Cashmere Goats

Shaanbei white cashmere（SBWC）goat is an excellent goat breed with both meat and cashmere,but its reproductive performance needs to be further improved.Therefore,it is important to use molecular marker-assisted selection（MAS）breeding for the genetic improvement of its reproductive traits.Based on others GWAS research result,a number of important genes（SMAD1,SMAD2,FSHR,CLSTN2,CXCL12,and CTNNB1）have been revealed can affect the reproductive traits.Using modern molecular genetics and molecular biology techniques,we first investigated the expression of six candidate genes in nine different tissues（the fallopian tube,kidney,lung,liver,fat,spleen,ovary,muscle,and the heart;n=15）and nine ovarian tissues from adult goat females with different reproductive abilities（mother of single kid;n=5;and mother of multiple kids;n=4）,followed by the detection of In Del（n=1390）and CNV（n=352;50 males and 302 females）variant loci,and the association between genetic variant loci with litter size and semen quality was analysed to investigate the influence of these loci on reproductive traits in large population of SBWC goats.This study will provide the theoretical basis and scientific guidance for the use of MAS breeding techniques to improve the reproductive traits of goats.1.Goat SMAD family member 1（SMAD1）:m RNA expression,genetic variants,and theirassociations with litter sizeSMAD family member 1（SMAD1）is phosphorylated and activated by the BMP receptors,which contributes to regulation ovulation rate,cell growth,apoptosis,and development.Previously,the genome-wide association study revealed that it has been associated with fecundity in sheep.However,there are no reports of In Del and CNV mutations in the SMAD1 gene being associated with litter size in goats.Therefore,the aim of this study was to investigate the expression of SMAD1 gene in various tissues of goats and ovaries of goats with different fertility,and to analyse the correlation between its polymorphisms（In Del and CNV）and litter size of SBWC.Homology analysis of the SMAD1 gene showed that the goat SMAD1 gene is conserved across species and has 100%homology with sheep,so it is hypothesized that the goat SMAD1 gene may perform a similar biological function to the sheep SMAD1 gene.As a result,q PCR analysis showed that SMAD1 was expressed in all nine tissues（n=15）in female SBWC goats,the highest levels in the fallopian tube,ovary,lung,fat,liver,and spleen（P<0.05）.Importantly,the m RNA expression level in the ovaries of multiple kids was higher than that of single kid（P<0.05）,presumably affecting reproductive traits.The In Del locus of the SMAD1 gene was then explored and its genetic effect was analysed in relation to litter size.Firstly,two In Dels（P1-Del-18-bp In Del and P2-Del-7-bp In Del）in intron 1 of SMAD1 were found polymorphic among ten potential primers in 1390 SBWC goats.Both P1-Del-18-bp In Del and P2-Del-7-bp In Del with ID genotypes had significantly higher correlations with litter size than other genotypes（P=0.014 and P=0.0001,respectively）.As shown by the chi-squared test,the genotypic distributions of 18-bp and 7-bp were significantly distinct between single-kid（P=0.02）and multiple kids（P=0.002）.Our findings confirm that two In Dels in SMAD1 were significantly associated with litter size and suggest that they could be used to improve litter size in goat breeding strategies.2.Investigation on m RNA expression and genetic variation within goat SMAD2 gene and itsassociation with reproduction（litter size and semen quality）The SMAD family member 2（SMAD2）belongs to the SMAD family,a member of the TGF-beta superfamily,which plays a significant role in the oogenesis and ovulation process.A genome-wide selective sweep analysis also found SMAD2 was associated with the fertility groups of Laoshan dairy goats,but its association with reproductive traits in goats is unclear.Based on this,this experiment was conducted to investigate the association between m RNA expression,In Del,and copy number variations of the SMAD2 gene with litter size in SBWC goats.In addition,the m RNA expression levels of SMAD2 were examined in various nine tissues of female goats and nine ovarian tissues from adult goat females with different reproductive stages.The results showed that SMAD2 was expressed in all nine tissues of goats,with high expression in the spleen and lung.Then,we further detected potential polymorphisms,including insertion/deletion and copy number variation,which were related to reproduction.Detection of the In Del mutation using six primer pairs revealed a polymorphism at a 12bp deletion site,and ANOVA showed that this locus was significantly associated with first-born litter size in SBWC goats（n=501,P=0.037）.The Chi-square（χ²）test revealed that the ID genotype was significantly more prevalent in mothers with multiple kids（P=0.01）,indicating that heterozygous individuals（ID）are more likely to produce multiple kids.By searching the Animal Omics Database,two CNV loci were found in the SMAD2 gene of goats.Identification and analysis of the two CNV mutations in the SMAD2 gene of SBWC goats revealed that both loci showed polymorphisms and were named CNV1 and CNV2,respectively.Correlation analysis showed that CNV2 was significantly associated with litter size in goats(P=3.59×10^-4),and CNV2 was also found to affect semen concentration（P<0.01）,ejaculate volume,live sperm count and sperm deformity rate（P<0.05）in male goats,with individuals of the Loss genotype outperforming those of other genotypes.Briefly,these findings suggest that the SMAD2 gene could be used as a candidate gene for litter size and semen quality in goat breeding through MAS.3.Expression profile,In Del,and CNV of the follicle-stimulating hormone receptor gene andits association with litter size in goatsFollicle-stimulating hormone receptor（FSHR）is a G protein-coupled receptor that contributes follicles grow,oocytes maturation,steroidogenesis,and granulosa cells growth.m RNA expression and genetic variation of the FSHR gene have not yet been demonstrated to affect litter size in SBWC goats.The goals of this study were to find out how the goat FSHR gene is expressed in m RNA as well as how insertion/deletion（In Del,n=1168）and copy number variations（CNV,n=120）affect litter size.In this study,the FSHR gene was found to be highly expressed in the ovaries and oviducts of goats;the m RNA expression levels of the FSHR gene in the ovaries of goats of different reproductive abilities were investigated and the results showed that the FSHR m RNA expression was significantly higher in MMK ovaries than in MSK（P<0.05）.The effect of In Del and CNV mutations in the FSHR gene on the litter size was analysed and only one In Del（17-bp-Del）mutation was found after In Del detection using 15 primer pairs and three potential CNVs were found after CNV detection using three primer pairs（CNV1-3）,and correlation analysis revealed that 17-bp-Del（P<0.01）,and 2 CNVs（CNV1;P<0.01;and CNV2;P<0.05）were significantly associated with litter size.For litter size,the ID genotype with the 17-bp-Del mutation was superior to the II genotype,and the Gain genotype with the CNV1 and CNV2 mutations was superior to the other genotypes.Overall,the 17 bp deletion in the FSHR gene and CNVs were found to be useful for MAS in goat breeding.4.Goat CLSTN2 gene:m RNA expression,In Del,CNVs and their associations with litter sizeCalsyntenin-2（CLSTN2）is a gene that encodes the synaptic protein calsyntenin 2,which is involved in cell proliferation,differentiation,cell death,tumorigenesis,and follicular expression.Although CLSTN2 has been identified as a potential candidate gene for sheep prolificacy,no studies have been done on its effect on goat prolificacy.The purpose of this study was to identify m RNA expression,In Del,and copy number variation within goat CLSTN2,and its association with litter size.Based on conservation of bioinformatics analysis,the phylogenetic tree and amino acid sequences of goat CLSTN2 gene had the highest homology with sheep.Herein,we uncovered significant differences in m RNA levels of the CLSTN2 gene in nine different tissues（n=15）in female goats（P<0.01）;it was highly expressed in the spleen,following ovary tissue.Nine primer pairs for In Del mutations and three primer pairs for CNV mutations were designed to investigate the correlation with litter size,but only one 16-bp deletion was discovered in female SBWC goats（n=902）,and three CNVs were found（n=118）.Association analysis revealed that the 16-bp deletion II genotype of the CLSTN2 gene（P<0.01）and the CNV2 Medium genotype（P<0.05）were the dominant genotypes significantly associated with litter size.The results of this study suggest that the CLSTN2 gene is a candidate gene that influences reproductive ability in goats and can be used for marker-assisted selection in goats.5.Detection of C-X-C motif chemokine ligand 12（CXCL12）gene:m RNA expression andgenetic polymorphism in Shaanbei white cashmere goatsCXC chemokine ligand 12（CXCL12）is a G protein-coupled receptor with a specific seventh transmembrane structure that plays an important role in early gestation and embryonic development.The aim of this study was to investigate the m RNA expression profile of the CXCL12 gene in various nine tissues of SBWC goats（each tissue,n=15）and the association between polymorphisms（n=352）with litter size in goats in order to gain a comprehensive understanding of CXCL12 gene expression and genetic effects in goats.In this work,amino acid sequence alignments and the phylogenetic tree of the goat CXCL12 gene were found to be highly conserved in sheep,cattle,and red deer.The results showed that CXCL12 was broadly expressed in nine different tissues（including ovarian tissue）.Interestingly,in the ovaries of goats of different fertility,CXCL12 gene expression was 4-fold higher in ovaries of fecundating multiple kids than in fecundating single-kids.In addition,four potential insertion/deletion（In Del）mutations were identified by PCR and one of the deletion mutations（16-bp-Del）was verified to be polymorphic after sequencing.Correlation analysis between genotype and litter size showed（n=352）that the DD genotype with this mutation produced more kids than the other genotypes（P<0.05）and that the mutation was distributed differently between single and multiple kidding groups（P<0.01）.These results tentatively explored the function of CXCL12 and may provide a theoretical reference for SBWC goat breeding.6.Exploring of CTNNB1 gene:expression pattern,polymorphism,CNV and associationanalysis with litter size in goatsThe Catenin beta 1（CTNNB1）gene is a member of theβ-catenin family.It is an important part of the canonical Wnt signaling pathway,initiated early in embryonic development,and expressed in the ovary.Therefore,the influence of the goat CTNNB1 gene and its genetic effect on the litter size of SBWC goats was examined.Further,bioinformatic analysis of the CTNNB1 gene showed that the protein was highly conserved in nine different species,including sheep.Quantitative real-time PCR（q PCR）was used to determine the expression profile of the CTNNB1 gene in the nine tissues（n=15）,which showed that the expression of CTNNB1 gene was significantly higher in ovaries,fallopian tube,lungs and kidneys（P<0.05）.At different reproductive stages,CTNNB1 gene expression was three times higher in ovarian tissues of multiple kids than in single kids.Further analysis of the CTNNB1gene In Del（n=706）with three potential primers revealed one polymorphic（18-bp deletion variant）and three CNVs were discovered by CNV analysis（n=120）.In the result,the 18 bp deletion with ID genotype and the CNV1 mutation with gain genotype had a significant effect on litter size（P<0.05）in the CTNNB1 gene.The above results suggest that the genetic effect of the CTNNB1 gene can be used as a molecular marker for goat breeding,which is helpful to accelerate the breeding of superior goat breeds.Overall,this study investigated and analyzed the expression of six candidate genes in various tissues of goats and their genetic variation in relation to litter size using molecular genetics and quantitative genetics and identified seven In Del mutations and 11 CNV mutations in six genes,including the SMAD1 gene P1-Del-18-bp and P2-Del-7-bp.The SMAD1 gene P1-Del-18-bp,P2-Del-7-bp locus,SMAD2 gene 12-bp deletion,CNV2 locus,FSHR gene 17-bp-Del,CNV1,CNV2 locus,CLSTN2 gene 16-bp deletion,CNV2 locus,CXCL12 gene 16-bp-Del locus,CTNNB1 gene 18-bp deletion,CNV1 locus all significantly affected the kidding number trait in goats.These markers can be used as effective molecular markers for kidding number in SBWC goats and provide some scientific and theoretical basis for practical breeding.