{"paper_id":"cb0a0354-b53c-43e4-836f-b7337e3f09ea","body_text":"Litter size is a very important trait affecting the economic benefits of the mutton sheep industry or farmers. However, the molecular mechanism of litter size in sheep has not been completely elucidated. The known major genes for litter size are very limited in sheep; therefore, the characterization of important genes and molecular markers for prolificacy is crucial for improving reproductive efficiency in sheep. By sequencing the whole genome of the 248 sheep from 36 landraces and 6 improved breeds around the world, Li et al. [ 1 ] reported key candidate genes for reproduction, including fibrillin-1  (FBN1 ), family with sequence similarity 184 member B  (FAM184B ) and zinc finger and AT-hook domain containing ( ZFAT ), combining the methods of genome-wide association analysis (GWAS) and selective signature analysis. This provides us with important candidate genes for further molecular characterization of the litter size of sheep.\nFBN1  has been proved to be associated with Marfan syndrome, polycystic ovary (POCS) and ovarian follicular development [ 2 , 3 , 4 , 5 ]. In recent years, it was discovered that  FBN1  may encode a novel protein, asprosin [ 6 ], which may be a novel regulator for ovarian follicular function [ 7 ]. Maylem et al. [ 7 ] verified the negative relationship between the abundance of  FBN1  mRNA and the diameters of bovine ovarian follicles. Using the GWAS method, the  FAM184B  gene has been screened and it was found that it was associated with the litter size of sheep, and was also highly expressed in the sheep follicle and hypothalamus [ 8 ]. Similarly, the gene had also been considered as a candidate gene for reproductive traits in Jinghai yellow chicken [ 9 ]. The  ZFAT  gene, strongly expressed in the placenta, was labeled as a new imprinted gene [ 10 ]. The expression of this gene is down-regulated in the placenta during pregnancy, especially in pathological placenta [ 10 ]. Kobayashi [ 11 ] found that the  ZFAT  gene was highly correlated with reproductive diseases such as endometriosis. However, the association between the above three genes ( FBN1 ,  FAM184B ,  ZFAT ) and the litter size of sheep has not been studied.\nTherefore, in this study, these three genes were selected as candidate genes to analyze their association with the litter size of polytocous and monotocous sheep. Firstly, based on our previous resequencing data of 10 sheep breeds [ 12 ], the polymorphic loci in these three genes were screened between the polytocous sheep group (Cele black sheep, Small Tail Han sheep, Hu sheep and Australian Merino sheep) and the monotocous sheep group (Prairie Tibetan sheep, Vally Tibetan sheep, Euler sheep, Bayanbulak sheep, Ujumqin sheep and Tan sheep). Then, the allele frequencies of each polymorphic locus in the 10 breeds and the population differentiation coefficient ( F st ) of each locus between the two groups were calculated. Next, the key candidate loci (g.160338382 T > C in  FBN1 , g.398531673 C > T in  FAM184B  and g.20150315 C > T in  ZFAT ) were selected for association analysis and if they met any one of the conditions: ① non-synonymous mutations with  F st  > 0.05, ② SNPs with  F st  > 0.15 in intron.  FecB  mutation is prevalent in many sheep breeds in China, and its effect is strong. In order to avoid  FecB  masking the effects of other genes and to analyze whether the effect of candidate loci is independent of  FecB  mutation, we analyzed the interaction effect between candidate loci and  FecB  on the litter size of ewes. This study will help to seek new genes and genetic markers associated with litter size for sheep breeding.\n\nBlood samples were collected from 751 ewes, which are from three polytocous breeds (Hu sheep, Cele black sheep, Xinggao mutton sheep) and two monotocous breeds (Sunite sheep, Bamei mutton sheep) ( Table 1 ). Xinggao mutton sheep is a new breed with high fertility and meat performance. Hu sheep and Cele black sheep are famous polytocous breeds in China. Both Sunite and Bamei mutton sheep are famous for high rate of meat production in China; however, they are monotocous breeds. DNA were extracted using TIANamp Genomic DNA kit (TIANGEN Co., Ltd., Beijing, China. DP304-03). The litter size information of Xinggao mutton sheep with the second and third parities was recorded.\nThe genotyping for the first three SNPs was conducted in a MassARRAY ®  SNP system. The genomic DNA of the samples was amplified using the amplification primers shown in  Table 2 . Then, the amplified products were digested with SAP enzyme. After that, the digested products were used as templates for extension reaction using the EXT primers ( Table 2 ). The single-base extended primers for three loci were designed via MassARRAY Assay Design v. 3.1 based on the sheep sequences of  FBN1, FAM184B  and  ZFAT  available in GenBank ARS-UI_Ramb_v2.0 (accession no.:  NC_056062.1 ,  NC_056060.1 ;  NC_056059.1 ). Finally, the extended products were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the genotypes of the SNP loci. Detailed information about the system and procedures has previously been described [ 13 , 14 ].  FecB  was amplified and genotyped according to our previous study [ 15 ].\nAllele and genotype frequency, polymorphism information content ( PIC ), heterozygosity ( He ) and number of effective alleles ( Ne ) were calculated using the following formulae: \n (1) \n P I C = 1 − ∑ i = 1 n p i 2 − ∑ i = 1 n − 1 ∑ j = i + 1 n 2 p i 2 p j 2 \n \n \n (2) \n H e = 1 − ∑ i = 1 n p i 2 \n \n \n (3) \n N e = 1 / ∑ i = 1 n p i 2 \n \nwhere  n  is the number of alleles, p i  is the allele frequency of the  i th allele and  p j  is the allele frequency of the  j th allele.\nChi-square test was used to detect whether the genotype distribution of each locus deviated from Hardy–Weinberg equilibrium. For the data that cannot be tested by chi-square test, we used the method of Fisher’s exact test to calculate the  P  value. The association of litter size with the genotypes of four SNPs was analyzed by SPSS 26 (ANOVA). The association of litter size with  FBN1  and  FecB  was analyzed using the following fixed effects model, with least squares means used for multiple comparisons of litter size among the different genotypes in Xinggao mutton sheep:  y  = μ  +  P  +  G1  +  G2  +  G1G2  +  e , where  y  is the phenotypic value of litter size,  μ  is the population mean,  P  is the fixed parity effect (two levels),  G1  is the fixed effect for the candidate SNPs,  G2  is the fixed effect for  FecB ,  G1G2  is the fixed interaction effect for the candidate SNPs and  FecB  and  e  is the random error effect of each observation. Analysis was performed using MASS package in R software (aov, Version 4.0.3) [ 16 ].\nFor the SNP significantly related to litter size, we further analyzed the location of the SNP and its influence on protein structure. Then, interaction network where the protein is located was constructed. If there is no existing protein network in sheep, it is necessary to construct a phylogenetic tree to determine the species whose protein network can be referenced. The candidate protein sequences of 11 species were aligned by CLUSTALW ( https://www.genome.jp/tools-bin/clustalw , accessed on 20 September 2023) and displayed by ESPript 3.0 ( https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi , accessed on 20 September 2023), and phylogenetic tree was constructed through the MEGA 11 and beautified on iToL ( https://itol.embl.de/ , accessed on 20 September 2023). The secondary structure of candidate protein was predicted using PredictProtein. The interaction network for candidate protein in model animals was predicted via STRING database v.12.0 [ 17 ] ( https://cn.string-db.org/ , accessed on 20 September 2023) and BioGRID database v.4.4 [ 18 ] ( https://thebiogrid.org/ , accessed on 20 September 2023), respectively.\n\nThe candidate SNPs (g.160338382 T > C in  FBN1 , g.398531673 C > T in  FAM184B , g.20150315 C > T in  ZFAT ) were genotyped in all ewe samples, and the genotyping results are shown in  Figure 1 . The results of population genetic analysis ( Table 3 ) indicated that the g.160338382 locus had moderate polymorphism (0.25 <  PIC  < 0.5) in most breeds except for Cele black sheep, the g.398531673 C > T locus had moderate polymorphism in Cele black sheep (0.25 <  PIC  < 0.5) and g.20150315 C > T had moderate polymorphism in Xinggao mutton sheep. The g.29382188 T > C locus had moderate polymorphism in Xinggao mutton sheep and Cele sheep (0.25 <  PIC  < 0.5). Hardy–Weinberg equilibrium tests revealed that g.160338382 T > C was under the Hardy–Weinberg equilibrium in five populations ( p  > 0.05), but the g.398531673 C > T locus was deviated from the Hardy–Weinberg equilibrium in Xinggao mutton sheep, Sunite sheep and Cele black sheep ( p  < 0.05).\nThe results of association analysis revealed that the g.160338382 T > C locus in  FBN1  was significantly associated with litter size in Xinggao mutton sheep ( Table 4 ). For this locus, the litter size in ewes with the CC genotype was significantly lower than that in ewes with the TT genotype in both the second parity and the third parity ( p  < 0.05). No significant association was observed between litter size and two loci in  FAM184B  and  ZFAT  genes. For the  FecB  mutation in  BMPR1B , the litter sizes of ewes with the GG genotypes were significantly higher than those of ewes with the AA genotype in both parities ( p  < 0.05). However, there was no significant interaction effect between  FecB  and the g.160338382 T > C locus on litter size ( Table 5 ,  Pr  > 0.05). Additionally, the litter size of Xinggao mutton sheep was significantly influenced by parity ( p  = 0.000019).\nBased on the results of association analysis, which showed that only  FBN1  g.160338382 T > C (except for  FecB ) had a significant relationship with litter size, we further checked the protein structure of FBN1 and analyzed the effect of this mutation on protein structure ( Figure 2 A). Interestingly, SNP g.160338382 T > C is adjacent to the anterior border of exon 58 ( Figure 2 B) and belongs to a splice polypyrimidine tract variant, which may lead to alternative splicing and ultimately cause changes in the structure and function of the protein. As shown in  Figure 2 A, exon 58 is located in the EGF-like domain, whose release is essential for inducing the ovulation process [ 19 ]. This suggests that the mutation might be closely related to ovulation.\nIn order to further understand the biological function of FBN1 in reproduction, we expect to obtain some valuable information from the protein interaction network. Since there is not an existing protein network for FBN1 protein in sheep, we first constructed a protein evolutionary tree ( Figure 3 A) to select the species that can be used for reference in protein network analysis. The result of the protein evolutionary tree displayed that two model animals ( Capra hircus  and  Bos taurus ) had the closest protein evolutionary relationship with sheep. In addition,  Homo sapiens  would also been involved in the protein interaction network analysis because there are many similarities in the reproduction mechanism between humans and sheep (bicorned uterus and similar ovulation rate) and in-depth function studies have been carried out on FBN1 protein in  Homo sapiens  [ 20 , 21 ]. Protein interaction network analysis for  Capra hircus, Homo sapiens  and  Bos taurus  demonstrated the 10 proteins most closely interacting with FBN1 ( Figure 3 B–D), and, among them, the five proteins (ELN, LUM, MFAP5, COL1A2, COL5A2) were associated with the proliferation of ovarian granulosa cells and cell markers for theca interna and granulosa cells [ 22 , 23 , 24 ]. Otherwise, the predicted protein network map of human FBN1 provided by the BioGRID database showed that KIAA1429, which is related with follicular development, has the high correlation with FBN1 besides the above interacting proteins ( Figure 3 E). These findings indicate that FBN1 plays an important role in follicular development together with the interacting proteins in these species.\n\nBy sequencing the whole genome of the 248 local sheep from 36 landraces and 6 improved breeds around the world, Li et al. [ 1 ] screened key candidate genes for reproduction, including  FBN1 ,  FAM184B  and  ZFAT , combining the methods of genome-wide association analysis (GWAS) and selective signature analysis. However, subsequent association analyses of these three genes with litter size have never been performed. Therefore, in this study, we first screened for polymorphic loci in these three genes based on our previous resequencing data of 10 sheep breeds and then determined the locus associated with litter size.\nPopulation genetic analysis indicated that the locus in  FAM184B  is not under the Hardy–Weinberg equilibrium in four breeds, including Xinggao mutton sheep, Cele black sheep, Sunite sheep and Bamei black sheep, suggesting that the locus may be subject to natural or artificial selection. In previous studies, the  FAM184B  gene was found in a candidate QTL region (close to Chr6:37.53 Mb) for meat traits in sheep and its eight SNPs have been identified as significant pleiotropic SNPs linked with meat traits [ 25 , 26 ]. Therefore, the  FAM184B  gene might be strongly selected during the domestication of the mutton sheep population. On the other hand, the other two loci of  FBN1  and  ZFAT  were both under the Hardy–Weinberg equilibrium in five breeds, so these two loci seem to have not been subjected to strong artificial selection. Moreover, as a famous polytocous locus, g.29382188 T > C in  BMPR1B  was deviated from the Hardy–Weinberg equilibrium in Hu sheep. This implies that this locus was subject to artificial selection during the domestication of Hu sheep in order to increase the litter size.\nIn this study, we analyzed the association between four loci and litter size with different parities. The results showed that  FBN1  g.160338382 T > C and  FecB  had a significant association with a litter size of second and third parity in Xinggao mutton sheep. As a major mutation of the high prolificacy of sheep,  FecB  was present in some Chinese prolific breeds of sheep [ 27 ], such as Hu sheep, Small Tail Han, Cele and Duolang sheep. In this study, we also found that  FecB  mutation was significantly correlated with litter size, but there was no significant interaction effect between  BMPR1B  and the  FBN1  g.160338382 T > C. This implies that the effect of  FBN1  g.160338382 T > C is obvious and independent of  FecB  on litter size.\nFBN1, a 350-KD glycoprotein, is the main component of microfibrils in the extracellular matrix [ 28 ]. As a member of the fibrillin protein family, FBN1 plays an indispensable role in fibrosis by regulating TGF-β signaling in the extracellular matrix [ 29 ]. Previous studies showed that fibroblasts or stromal cells are the major cell type present in the stroma of many organs [ 30 ] and can result in defects in the function of organs, such as polycystic ovary syndrome (POCS) [ 31 ]. By measuring the mRNA abundance of FBN1 protein in the granulosa cell of  Bovine  and  Buffalo  ovaries, the researchers found that, in both species, the  FBN1  mRNA abundance in small follicles was significantly greater than that in large follicles [ 7 , 32 ]. Furthermore, FBN1 plays an important role in follicular development [ 5 ] and granulosa cell proliferation [ 32 ].\nInterestingly, as an intron mutation,  FBN1  g.160338382 T > C might affect the splicing and transcription of exons 58 of FBN1. As a polypeptide protein translated from 65 exons, FBN1 protein is encoded by 2876 amino acids. Among them, amino acids 1–24 encode signal peptides and amino acids 25–2876 encode protein binding (EGF-like domain (EGF-like calcium-binding domain)), DNA binding, RNA binding and a small segment of a disordered region. Exon 58 (ENSOARE00000208277) is encoded by 42 amino acids and is located in the EGF-like domain, including DNA binding and RNA binding. Previous studies showed that the release of the EGF-like domain by a specific enzyme is essential for interaction with the EGFR for granulosa cell luteinization, cumulus expansion and oocyte maturation [ 19 , 33 ]. EGF-like domains of exon 58 are 45–60-amino-acid-long domains located between the transmembrane domains and the mucin domains. In recent years, researchers have found that adding EGF to cumulus cells cultured in vitro can phosphorylate the receptors (ERK1/2, EGFR) in the cells and activate the expansion and the maturation of oocytes of  Sus scrofa  [ 34 ],  Mouse  [ 35 ] and  Bovine  ovaries [ 36 ]. During ovulation, multiple signaling pathways in cumulus cells and granulosa cells are activated. EGF-like factors are responsible for the RAS-cRAF-MEK1-ERK1/2 pathway in spermatogonia and granulosa cells [ 37 , 38 ]. Previous studies have shown that the addition of EGFR inhibitors or specific knockout of EGF domain coding genes in follicular cells can cause the expansion of the cumulus to become dramatically blocked [ 39 , 40 , 41 ]. They also clearly revealed that the release of the EGF domain acted on EGFR in both granulosa cells and cumulus cells to activate the ERK1/2 pathway [ 42 ]. Coincidentally, this mutation is adjacent to the anterior border of exon 58 ( Figure 2 B) and belongs to a splice polypyrimidine tract variant, which may lead to alternative splicing and changes in the structure of protein and may ultimately affect the proliferation and cumulus expansion of granulosa cells and ovulation.\nIn addition, FBN1 protein had been shown to play an important role in cell differentiation and apoptosis in different cell types and species [ 5 , 43 ]. FBN1 stimulates follicular atresia in the presence of BMP15 (a ligand that can stimulate follicular development) based on its effects on cumulus cell apoptosis. In the presence of BMP15, FBN1 depletion protected cumulus cells from apoptosis. Similarly, when  FBN1  was depleted, the addition of BMP15 played a protective role for cumulus cells [ 44 ]. Thus, we summarized that FBN1 is an important regulatory factor for BMP15 to inhibit the apoptosis of porcine ovarian cumulus cells. When the mutation causes abnormal transcription, it will promote the apoptosis of granulosa cells and reduce the number of ovulations. Therefore, this may be the reason why we observed a decrease in litter size in mutant ewes in this study.\nThe results of the protein interaction network showed that the six proteins that have the closest relationship with FBN1 are associated with follicular development and ovulation. For interacting proteins KIAA1249, previous studies have shown that the specific deletion of KIAA1249 in mouse oocytes leads to defects in follicular development, loss of the meiotic performance of oocytes and ultimately female infertility [ 45 ]. The five proteins (ELN, LUM, MFAP5, COL1A2 and COL5A2) were highlighted from the protein network provided by the STRING. ELN was identified as a gene significantly related to chicken follicular development by transcriptome profiling analysis [ 46 ]. LUM has been reported to be involved in ovulation in  Homo sapiens  and bovine [ 47 , 48 ]. MFAP5 was related to ovarian hyperplasia and endometrial growth [ 49 , 50 ]. As the main component of extracellular matrix (ECM) protein in the mammalian ovary, COL1A2 plays a vital role in follicular development and corpus luteum formation [ 22 , 51 , 52 ]. In summary, the FBN1 protein forms a network with the above proteins to coordinately regulate follicular development and ovulation.\n\nThe present study revealed that an SNP (g.160338382 T > C) in  FBN1  is significantly associated with litter size in Xinggao mutton sheep. Moreover, this effect is independent of the  FecB  mutation. Therefore, the locus might be considered as a potentially effective genetic marker for improving litter size.","source_license":"CC-BY-4.0","license_restricted":false}