Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system.

BACKGROUND: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips. METHODS: We firstly delivered a 13 kb Cas9-EGFP and a 3.5 kb pMAX-GFP vector into PFFs via 7 programs recommended by the Lonza basic protocol. We then tested 6 customized dual-electroporation programs for delivering the 13 kb plasmid into PFFs. In addition, we screened potential alternative electroporation buffers to the Nucleofector™ P3 solution. Finally, three CRISPR/Cas9-sgRNAs targeting Rosa26, H11, and Cep112 loci were delivered into PFFs with different single and dual-electroporation programs. RESULTS: Notably lower transfection efficiencies were observed when delivering the 13 kb vector than delivering the 3.5 kb vector in PFFs via the single-electroporation programs. The customized dual-electroporation program FF-113 + CA-137 exhibited higher transfection efficiencies than any of the single-electroporation programs using vessels (98.1%) or strips (89.1%) with acceptable survival rates for the 13 kb vector. Entranster-E buffer generated similar transfection efficiencies and 24-hour survival rates to those from the P3 solution, thus can be used as an alternative electroporation buffer. In the genome-editing experiments, the FF-113 + CA-137 and CA-137 + CA-137 programs showed significantly superior (P < 0.01) efficiencies to ones from the single-electroporation programs in vessels and strips. Entranster-E buffer produced higher indel efficiencies than the P3 buffer. CONCLUSIONS: We markedly increased the delivery efficiencies for a large vector via customized dual-electroporation programs using Lonza 4D-Nucleofector™ system, and Entranster-E buffer can be used as an alternative electroporation buffer to Nucleofector™ P3 buffer.

Highly Efficient A-to-G Editing in PFFs via Multiple ABEs.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In this study, five ABEs, ABE7.10, ABEmax, NG-ABEmax, ABE8e and NG-ABE8e, were used to generate A-to-G (T-to-C) conversions in five genome loci in porcine fetal fibroblasts (PFFs). Variable yet appreciable editing efficiencies and variable activity windows were observed in these targeting regions via these five editors. The strategy of two sgRNAs in one vector exhibited superior editing efficiency to that of using two separate sgRNA expression vectors. ABE-mediated start-codon mutation in APOE silenced its expression of protein and, unexpectedly, eliminated the vast majority of its mRNA. No off-target DNA site was detected for these editors. Substantial off-target RNA events were present in the ABE-edited cells, but no KEGG pathway was found to be significantly enriched. Our study supports that ABEs are powerful tools for A-to-G (T-to-C) point-mutation modification in porcine cells.

Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor.

Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes. C−to−T conversion was found in 98.6%, 92.8%, and 87.2% of these cell colonies for TP53, PTEN, and APC, respectively. High frequencies of bystander C−to−T edits were observed within the editing window (positions 3−8), and there were nine (3.01%) clones with the designed simultaneous three-gene C−to−T conversion without bystander conversion. C−to−T conversion outside the editing window was found in 9.0%, 14.1%, and 26.2% of the 290 cell colonies for TP53, PTEN, and APC, respectively. Low-frequency C−to−G or C−to−A transversion occurred in APC. The mRNA levels of the three genes showed significant declines in triple-gene-mutant (Tri-Mut) cells as expected. No PTEN and a significantly lower (p < 0.05) APC protein expression were detected in Tri-Mut cells. Interestingly, the premature stop codon introduced into the TP53 gene did not eliminate the expression of its full-length protein in the Tri-Mut cells, suggesting that stop codon read-through occurred. Tri-Mut cells showed a significantly higher (p < 0.05) proliferation rate than WT cells. Furthermore, we identified 1418 differentially expressed genes (DEGs) between the Tri-Mut and WT groups, which were mainly involved in functions such as tumor progression, cell cycle, and DNA repair. This study indicates that hA3A-BE3-Y130F can be a powerful tool to create diverse knockout cell models without double-strand breaks (DSBs), with further possibilities to produce porcine models with various purposes.

BMPR-IB gene disruption causes severe limb deformities in pigs.

In an attempt to generate g.A746G substitution in the BMPR-IB gene, we unexpectedly obtained BMPR-IB homozygous knockout piglets ( BMPR-IB -/-) and heterogeneous knockout piglets with one copy of the A746G mutation ( BMPR-IB -/746G) via CRISPR/Cas9 editing. Polymerase chain reaction (PCR) and sequencing revealed complex genomic rearrangements in the target region. All BMPR-IB-disrupted piglets showed an inability to stand and walk normally. Both BMPR-IB -/- and BMPR-IB -/746G piglets exhibited severe skeletal dysplasia characterized by distorted and truncated forearms (ulna, radius) and disordered carpal, metacarpal, and phalangeal bones in the forelimbs. The piglets displayed more severe deformities in the hindlimbs by visual inspection, including fibular hemimelia, enlarged tarsal bone, and disordered toe joint bones. Limb deformities were more profound in BMPR-IB -/- piglets than in the BMPR-IB -/746G piglets. Proteomic analysis identified 139 differentially expressed proteins (DEPs) in the hindlimb fibula of BMPR -IB -/746G piglets compared to the wild-type (WT) controls. Most DEPs are involved in skeletal or embryonic development and/or the TGF-ß pathway and tumor progression. Gene Ontology (GO) and protein domain enrichment analysis suggested alterations in these processes. Of the top 50 DEPs, a large proportion, e.g., C1QA, MYO1H, SRSF1, P3H1, GJA1, TCOF1, RBM10, SPP2, MMP13, and PHAX, were significantly associated with skeletal development. Our study provides novel findings on the role of BMPR-IB in mammalian limb development.

An Integrative Analysis of Transcriptome and GWAS Data to Identify Potential Candidate Genes Influencing Meat Quality Traits in Pigs.

Understanding the genetic factors behind meat quality traits is of great significance to animal breeding and production. We previously conducted a genome-wide association study (GWAS) for meat quality traits in a White Duroc × Erhualian F2 pig population using Illumina porcine 60K SNP data. Here, we further investigate the functional candidate genes and their network modules associated with meat quality traits by integrating transcriptomics and GWAS information. Quantitative trait transcript (QTT) analysis, gene expression QTL (eQTL) mapping, and weighted gene co-expression network analysis (WGCNA) were performed using the digital gene expression (DGE) data from 493 F2 pig's muscle and liver samples. Among the quantified 20,108 liver and 23,728 muscle transcripts, 535 liver and 1,014 muscle QTTs corresponding to 416 and 721 genes, respectively, were found to be significantly (p < 5 × 10-4) correlated with 22 meat quality traits measured on longissiums dorsi muscle (LM) or semimembranosus muscle (SM). Transcripts associated with muscle glycolytic potential (GP) and pH values were enriched for genes involved in metabolic process. There were 42 QTTs (for 32 genes) shared by liver and muscle tissues, of which 10 QTTs represent GP- and/or pH-related genes, such as JUNB, ATF3, and PPP1R3B. Furthermore, a genome-wide eQTL mapping revealed a total of 3,054 eQTLs for all annotated transcripts in muscle (p < 2.08 × 10-5), including 1,283 cis-eQTLs and 1771 trans-eQTLs. In addition, WGCNA identified five modules relevant to glycogen metabolism pathway and highlighted the connections between variations in meat quality traits and genes involved in energy process. Integrative analysis of GWAS loci, eQTL, and QTT demonstrated GALNT15/GALNTL2 and HTATIP2 as strong candidate genes for drip loss and pH drop from postmortem 45 min to 24 h, respectively. Our findings provide valuable insights into the genetic basis of meat quality traits and greatly expand the number of candidate genes that may be valuable for future functional analysis and genetic improvement of meat quality.

Integrated analysis of miRNA and mRNA expression profiles in p53-edited PFF cells.

p53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells. Totally 28 annotated DEMs were found to overlap between p53-KO/p53-WT and p53-241W242S/p53-WT miRNAs datasets, of which miR-34 c, miR-218, miR-205, miR-105-1, miR-105-2, miR-206, miR-224 and miR-429 play important roles in p53 regulatory network. Among the top 10 DEGs in p53-KO and p53-241W242S cells, most genes were reported to be involved in tumors, cell proliferation or cell migration. p53-KO and p53-241W242S cells showed a significantly higher (P < 0.01) proliferation rate compared with p53-WT cells. In conclusion, genetic modifications of p53 gene significantly affect the expression levels of a large number of genes and miRNAs in the PFF cells. The p53-edited PFF cells could be used as non-tumor cell models for investigating the p53 signaling network, and as donor cells for somatic nuclear transfer, with the aim to develop porcine models with the corresponding p53 mutations.Abbreviations: CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; PFF: porcine fetal fibroblasts; SCNT: somatic cell nuclear transfer; RNA sequencing: small RNA sequencing and mRNA sequencing; DEGs: differentially expressed mRNAs; DEMs: differentially expressed miRNAs.

Semen quality improvement in boars fed with supplemental wolfberry (Lycium barbarum).

Wolfberry is well known for its health benefits in Asian countries. This study consisted of two experiments. In Experiment 1, nine boars were provided 40 g dried wolfberry per 100 kg body weight per day in addition to regular feed for 160 days (divided into 40 days phases: I, II, III, and IV) under step-down air temperature conditions. Controls (n = 9) were fed regular feed only. Significant (p < .05 or p < .01) or slight improvements in sperm progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate were observed in the wolfberry group during phases II and III. No differences were observed in semen volume. After combining the data from phases II ~ IV, significant improvements were detected in all aforementioned traits (p < .05 or p < .01), except semen volume. In Experiment 2, the wolfberry group (n = 5) was fed wolfberry for 90 days and exhibited significantly reduced head, tail, and total abnormality rates (p < .05 or p < .01) in both fresh semen and semen stored for 72 hr at 17°C compared to the control group (n = 5). SOD activity also significantly increased in this group of boars. Collectively, the findings of this study suggest that wolfberry has a positive effect on boar semen quality.

A model of mucopolysaccharidosis type IIIB in pigs.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare genetic disorder caused by loss-of-function mutations in the NAGLU gene. Pigs are an ideal large animal model for human diseases; however, a porcine model of MPS IIIB has not been reported. We have previously generated a heterozygous NAGLU-deficient (NAGLU +/-) Large White boar via a transgenic approach. Here we characterized phenotypes of the F1 offspring of this founder to establish a pig model for MPS IIIB. qRT-PCR revealed that the NAGLU expression level was significantly decreased in a variety of tissues in NAGLU +/- pigs. ELISA assays showed obvious deficiency of NAGLU and higher (P<0.05) glycosaminoglycan levels in multiple tissues from NAGLU +/- pigs. NAGLU +/- pigs grew at a significantly (P<0.05) slower rate than control animals (NAGLU +/+). Death, mostly sudden death, occurred at all ages in NAGLU +/- pigs, most of which died within two years. Necropsy findings included pleural adhesions, lung shrinkage and abnormalities in the pericardium and mild hepatomegaly in NAGLU +/- pigs. Notable pathological changes were observed in the sections of brain, liver, spleen and kidney from NAGLU +/- pigs. Brain atrophy, ventriculomegaly, cerebellar atrophy and abnormalities in the intracerebral capsule, parietal lobes and the thalamus were also evident in NAGLU +/- pigs. Together, NAGLU +/- pigs show typical symptoms of human MPS IIIB patients and thus represent a novel large animal model for the disease.This article has an associated First Person interview with the first author of the paper.

VRTN is Required for the Development of Thoracic Vertebrae in Mammals.

Vertnin (VRTN) variants are associated with thoracic vertebral number (TVN) in pigs. However, the biological function of VRTN remains poorly understood. Here we first conducted a range of experiments to demonstrate that VRTN is a responsible gene for TVN and two causative variants in the regulatory region of VRTN additively regulate TVN. Then, we show that VRTN is a novel DNA-binding transcription factor as it localizes exclusively in the nucleus, binds to DNA on a genome-wide scale and regulates the transcription of a set of genes that harbor VRTN binding motifs. Next, we illustrate that VRTN is essential for the development of thoracic vertebrae. Vrtn-null embryos display somitogenesis defect with the failure of axial rotation and fewer somites at the thoracic somite stage. Half of Vrtn heterozygous mice show abnormal spinal development with fewer thoracic vertebrae and ribs than their wild-type littermates. Lastly, we reveal that VRTN could modulate somite segmentation via the Notch signaling pathway. The findings advance our understanding of the mechanisms underlying the development of thoracic vertebrate in mammals, and VRTN causative variants provide a robust tool to improve pork production by selecting the alleles increasing the number of thoracic vertebrae and ribs.

The increased expression of follicle-stimulating hormone leads to a decrease of fecundity in transgenic Large White female pigs.

Follicle-stimulating hormone (FSH) is a pituitary gonadotropin regulating reproduction in mammals. Overexpression of the exogenous FSHα/ß genes from Chinese Erhualian pigs improved female fecundity of transgenic (TG) mice and male spermatogenesis ability of Large White TG boars. Here, we investigated the impact of the exogenous FSHα/ß genes on female reproductive performance of Large White TG pigs. First, we identified the integration site of the exogenous FSHα/ß genes at 140,646,456 bp on chromosome 9 in these TG pigs using whole-genome sequencing. Then, we showed that TG gilts had higher levels of serum FSH and FSHß protein in pituitary while had a potentially lower number of born piglets than their wild-type half sibs. TG gilts grew healthily and normally without significant difference in growth and health parameters as compared to WT gilts. The expression levels of FSHR, LHR, ESR1 and ESR2 were significantly lower in TG gilts than in WT gilts at the age of 300 days. Taken together, we proposed that the overexpressed FSHα/ß transgenes could cause deteriorate fecundity via disturbing the normal expression of the endogenous reproduction-related genes in female pigs. Our findings provide insight into the effect of overexpression of FSHα/ß on female reproduction performance in pigs.

BAC mediated transgenic Large White boars with FSHα/ß genes from Chinese Erhualian pigs.

Follicle-stimulating hormone (FSH) is a critical hormone regulating reproduction in mammals. Transgenic mice show that overexpression of FSH can improve female fecundity. Using a bacterial artificial chromosome (BAC) system and somatic cell nuclear transfer, we herein generated 67 Large White transgenic (TG) boars harboring FSHα/ß genes from Chinese Erhualian pigs, the most prolific breed in the world. We selected two F0 TG boars for further breeding and conducted molecular characterization and biosafety assessment for F1 boars. We showed that 8-9 copies of exogenous FSHα and 5-6 copies of exogenous FSHß were integrated into the genome of transgenic pigs. The inheritance of exogenous genes conforms to the Mendel's law of segregation. TG boars had higher levels of serum FSH, FSHα mRNA in multiple tissues, FSHß protein in pituitary and more germ cells per seminiferous tubule compared with their wild-type half sibs without any reproductive defects. Analysis of growth curve, hematological and biochemical parameters and histopathology illustrated that TG boars grew healthily and normally. By applying 16S rRNA gene sequencing, we demonstrated that exogenous genes had no impact on the bacterial community structures of pig guts. Moreover, foreign gene drift did not occur as verified by horizontal gene transfer. Our findings indicate that overexpression of FSH could improve spermatogenesis ability of boars. This work provides insight into the effect of FSHα/ß genes on male reproductive performance on pigs by a BAC-mediated transgenic approach.

Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy.

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

[Application of CRISPR/Cas9 mediated genome editing in farm animals].

CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) is an acquired immune system found in bacteria and archaea that fight against invasion of viruses or plasmids. CRISPR/Cas systems are currently classified into three main types: I, II and III, of which type II has relatively simple components. The CRISPR/Cas9 technology modified from type II CRISPR/Cas system has been developed as an efficient genome editing tool. Since the initial application of the CRISPR/Cas9 technology in mammals in 2013, the reports of this system for genomic editing has skyrocketed. Farm animals are not only economically important animals, but also ideal animal models for human diseases and biomedical studies. In this review, we summarize the applications of CRISPR/Cas9 in farm animals, briefly describe the off-target effects and the main solutions, and finally highlight the future perspectives of this technology.

An imputation-based genome-wide association study on traits related to male reproduction in a White Duroc × Erhualian F2 population.

Boar reproductive traits are economically important for the pig industry. Here we conducted a genome-wide association study (GWAS) for 13 reproductive traits measured on 205 F2 boars at day 300 using 60 K single nucleotide polymorphism (SNP) data imputed from a reference panel of 1200 pigs in a White Duroc × Erhualian F2 intercross population. We identified 10 significant loci for seven traits on eight pig chromosomes (SSC). Two loci surpassed the genome-wide significance level, including one for epididymal weight around 60.25 Mb on SSC7 and one for semen temperature around 43.69 Mb on SSC4. Four of the 10 significant loci that we identified were consistent with previously reported quantitative trait loci for boar reproduction traits. We highlighted several interesting candidate genes at these loci, including APN, TEP1, PARP2, SPINK1 and PDE1C. To evaluate the imputation accuracy, we further genotyped nine GWAS top SNPs using PCR restriction fragment length polymorphism or Sanger sequencing. We found an average of 91.44% of genotype concordance, 95.36% of allelic concordance and 0.85 of r(2) correlation between imputed and real genotype data. This indicates that our GWAS mapping results based on imputed SNP data are reliable, providing insights into the genetic basis of boar reproductive traits.

Porcine SOX9 Gene Expression Is Influenced by an 18 bp Indel in the 5'-Untranslated Region.

Sex determining region Y-box 9 (SOX9) is an important regulator of sex and skeletal development and is expressed in a variety of embryonal and adult tissues. Loss or gain of function resulting from mutations within the coding region or chromosomal aberrations of the SOX9 locus lead to a plethora of detrimental phenotypes in humans and animals. One of these phenotypes is the so-called male-to-female or female-to-male sex-reversal which has been observed in several mammals including pig, dog, cat, goat, horse, and deer. In 38,XX sex-reversal French Large White pigs, a genome-wide association study suggested SOX9 as the causal gene, although no functional mutations were identified in affected animals. However, besides others an 18 bp indel had been detected in the 5'-untranslated region of the SOX9 gene by comparing affected animals and controls. We have identified the same indel (Δ18) between position +247 bp and +266 bp downstream the transcription start site of the porcine SOX9 gene in four other pig breeds; i.e., German Large White, Laiwu Black, Bamei, and Erhualian. These animals have been genotyped in an attempt to identify candidate genes for porcine inguinal and/or scrotal hernia. Because the 18 bp segment in the wild type 5'-UTR harbours a highly conserved cAMP-response element (CRE) half-site, we analysed its role in SOX9 expression in vitro. Competition and immunodepletion electromobility shift assays demonstrate that the CRE half-site is specifically recognized by CREB. Both binding of CREB to the wild type as well as the absence of the CRE half-site in Δ18 reduced expression efficiency in HEK293T, PK-15, and ATDC5 cells significantly. Transfection experiments of wild type and Δ18 SOX9 promoter luciferase constructs show a significant reduction of RNA and protein levels depending on the presence or absence of the 18 bp segment. Hence, the data presented here demonstrate that the 18 bp indel in the porcine SOX9 5'-UTR is of functional importance and may therefore indeed be a causative variation in SOX9 associated traits.

Understanding the molecular mechanisms of human microtia via a pig model of HOXA1 syndrome.

Microtia is a congenital malformation of the outer ears. Although both genetic and environmental components have been implicated in microtia, the genetic causes of this innate disorder are poorly understood. Pigs have naturally occurring diseases comparable to those in humans, providing exceptional opportunity to dissect the molecular mechanism of human inherited diseases. Here we first demonstrated that a truncating mutation in HOXA1 causes a monogenic disorder of microtia in pigs. We further performed RNA sequencing (RNA-Seq) analysis on affected and healthy pig embryos (day 14.25). We identified a list of 337 differentially expressed genes (DEGs) between the normal and mutant samples, shedding light on the transcriptional network involving HOXA1. The DEGs are enriched in biological processes related to cardiovascular system and embryonic development, and neurological, renal and urological diseases. Aberrant expressions of many DEGs have been implicated in human innate deformities corresponding to microtia-associated syndromes. After applying three prioritizing algorithms, we highlighted appealing candidate genes for human microtia from the 337 DEGs. We searched for coding variants of functional significance within six candidate genes in 147 microtia-affected individuals. Of note, we identified one EVC2 non-synonymous mutation (p.Asp1174Asn) as a potential disease-implicating variant for a human microtia-associated syndrome. The findings advance our understanding of the molecular mechanisms underlying human microtia, and provide an interesting example of the characterization of human disease-predisposing variants using pig models.

A splice mutation in the PHKG1 gene causes high glycogen content and low meat quality in pig skeletal muscle.

Glycolytic potential (GP) in skeletal muscle is economically important in the pig industry because of its effect on pork processing yield. We have previously mapped a major quantitative trait loci (QTL) for GP on chromosome 3 in a White Duroc × Erhualian F2 intercross. We herein performed a systems genetic analysis to identify the causal variant underlying the phenotype QTL (pQTL). We first conducted genome-wide association analyses in the F2 intercross and an F19 Sutai pig population. The QTL was then refined to an 180-kb interval based on the 2-LOD drop method. We then performed expression QTL (eQTL) mapping using muscle transcriptome data from 497 F2 animals. Within the QTL interval, only one gene (PHKG1) has a cis-eQTL that was colocolizated with pQTL peaked at the same SNP. The PHKG1 gene encodes a catalytic subunit of the phosphorylase kinase (PhK), which functions in the cascade activation of glycogen breakdown. Deep sequencing of PHKG1 revealed a point mutation (C>A) in a splice acceptor site of intron 9, resulting in a 32-bp deletion in the open reading frame and generating a premature stop codon. The aberrant transcript induces nonsense-mediated decay, leading to lower protein level and weaker enzymatic activity in affected animals. The mutation causes an increase of 43% in GP and a decrease of>20% in water-holding capacity of pork. These effects were consistent across the F2 and Sutai populations, as well as Duroc × (Landrace × Yorkshire) hybrid pigs. The unfavorable allele exists predominantly in Duroc-derived pigs. The findings provide new insights into understanding risk factors affecting glucose metabolism, and would greatly contribute to the genetic improvement of meat quality in Duroc related pigs.

A further look at porcine chromosome 7 reveals VRTN variants associated with vertebral number in Chinese and Western pigs.

The number of vertebrae is an economically important trait that affects carcass length and meat production in pigs. A major quantitative trait locus (QTL) for thoracic vertebral number has been repeatedly identified on pig chromosome (SSC) 7. To dissect the genetic basis of the major locus, we herein genotyped a large sample of animals from 3 experimental populations of Chinese and Western origins using 60K DNA chips. Genome-wide association studies consistently identified the locus across the 3 populations and mapped the locus to a 947-Kb region on SSC7. An identical-by-descent sharing assay refined the locus to a 100-Kb segment that harbors only two genes including VRTN and SYNDIG1L. Of them, VRNT has been proposed as a strong candidate of the major locus in Western modern breeds. Further, we resequenced the VRTN gene using DNA samples of 35 parental animals with known QTL genotypes by progeny testing. Concordance tests revealed 4 candidate causal variants as their genotypes showed the perfect segregation with QTL genotypes of the tested animals. An integrative analysis of evolutional constraints and functional elements supported two VRTN variants in a complete linkage disequilibrium phase as the most likely causal mutations. The promising variants significantly affect the number of thoracic vertebrae (one vertebra) in large scale outbred animals, and are segregating at rather high frequencies in Western pigs and at relatively low frequencies in a number of Chinese breeds. Altogether, we show that VRTN variants are significantly associated with the number of thoracic vertebrae in both Chinese and Western pigs. The finding advances our understanding of the genetic architecture of the vertebral number in pigs. Furthermore, our finding is of economical importance as it provides a robust breeding tool for the improvement of vertebral number and meat production in both Chinese indigenous pigs and Western present-day commercial pigs.

A whole genome scanning for quantitative trait loci on traits related to sperm quality and ejaculation in pigs.

To identify quantitative trait loci (QTL) for traits related to semen and ejaculation, phenotype data including semen volume, sperm concentration, total sperm per ejaculate, sperm motility, sperm abnormality rate, semen pH value, ejaculation times and ejaculation duration were measured on 206 F(2) boar at 240 days in a White Duroc x Erhualian intercross. A genome-wide scan was performed and the entire White Duroc x Erhualian intercross was genotyped for 183 microsatellite markers covering the whole pig genome. QTL analysis was performed using a composite regression interval mapping method via QTLExpress. A total of 18 QTL were detected, including 4 genome-wide significant QTL each for semen pH on pig chromosome (SSC) 2 and SSC12, for semen volume on SSC15, and for ejaculation times on SSC17. Fourteen suggestive QTL were found on SSC1, 2, 3, 4, 6, 9, 17 and 18. To our knowledge, this is the first report about the QTL for semen and ejaculation traits in pigs, providing a start point to decipher the genetic basis of these complex traits.