Mapping and functional characterization of structural variation in 1060 pig genomes.

BACKGROUND: Structural variations (SVs) have significant impacts on complex phenotypes by rearranging large amounts of DNA sequence. RESULTS: We present a comprehensive SV catalog based on the whole-genome sequence of 1060 pigs (Sus scrofa) representing 101 breeds, covering 9.6% of the pig genome. This catalog includes 42,487 deletions, 37,913 mobile element insertions, 3308 duplications, 1664 inversions, and 45,184 break ends. Estimates of breed ancestry and hybridization using genotyped SVs align well with those from single nucleotide polymorphisms. Geographically stratified deletions are observed, along with known duplications of the KIT gene, responsible for white coat color in European pigs. Additionally, we identify a recent SINE element insertion in MYO5A transcripts of European pigs, potentially influencing alternative splicing patterns and coat color alterations. Furthermore, a Yorkshire-specific copy number gain within ABCG2 is found, impacting chromatin interactions and gene expression across multiple tissues over a stretch of genomic region of ~200 kb. Preliminary investigations into SV's impact on gene expression and traits using the Pig Genotype-Tissue Expression (PigGTEx) data reveal SV associations with regulatory variants and gene-trait pairs. For instance, a 51-bp deletion is linked to the lead eQTL of the lipid metabolism regulating gene FADS3, whose expression in embryo may affect loin muscle area, as revealed by our transcriptome-wide association studies. CONCLUSIONS: This SV catalog serves as a valuable resource for studying diversity, evolutionary history, and functional shaping of the pig genome by processes like domestication, trait-based breeding, and adaptive evolution.

Evaluating three strategies of genome-wide association analysis for integrating data from multiple populations.

In livestock, genome-wide association studies (GWAS) are usually conducted in a single population (single-GWAS) with limited sample size and detection power. To enhance the detection power of GWAS, meta-analysis of GWAS (meta-GWAS) and mega-analysis of GWAS (mega-GWAS) have been proposed to integrate data from multiple populations at the level of summary statistics or individual data, respectively. However, there is a lack of comparison for these different strategies, which makes it difficult to guide the best practice of GWAS integrating data from multiple study populations. To maximize the comparison of different association analysis strategies across multiple populations, we conducted single-GWAS, meta-GWAS, and mega-GWAS for the backfat thickness of 100 kg (BFT_100) and days to 100 kg (DAYS_100) within each of the three commercial pig breeds (Duroc, Yorkshire, and Landrace). Based on controlling the genome inflation factor to one, we calculated corrected p-values (pC ). In Yorkshire, with the largest sample size, mega-GWAS, meta-GWAS and single-GWAS detected 149, 38 and 20 significant SNPs (pC < 1E-5) associated with BFT_100, as well as 26, four, and one QTL, respectively. Among them, pC of SNPs from mega-GWAS was the lowest, followed by meta-GWAS and single-GWAS. The correlation of pC among the three GWAS strategies ranged from 0.60 to 0.75 and the correlation of SNP effect values between meta-GWAS and mega-GWAS was 0.74, all showing good agreement. Collectively, even though there are differences in the integration of individual data or summary statistics, integrating data from multiple populations is an effective means of genetic argument for complex traits, especially mega-GWAS versus single-GWAS.

A compendium of genetic regulatory effects across pig tissues.

The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model.

PigBiobank: a valuable resource for understanding genetic and biological mechanisms of diverse complex traits in pigs.

To fully unlock the potential of pigs as both agricultural species for animal-based protein food and biomedical models for human biology and disease, a comprehensive understanding of molecular and cellular mechanisms underlying various complex phenotypes in pigs and how the findings can be translated to other species, especially humans, are urgently needed. Here, within the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we build the PigBiobank (http://pigbiobank.farmgtex.org) to systematically investigate the relationships among genomic variants, regulatory elements, genes, molecular networks, tissues and complex traits in pigs. This first version of the PigBiobank curates 71 885 pigs with both genotypes and phenotypes from over 100 pig breeds worldwide, covering 264 distinct complex traits. The PigBiobank has the following functions: (i) imputed sequence-based genotype-phenotype associations via a standardized and uniform pipeline, (ii) molecular and cellular mechanisms underlying trait-associations via integrating multi-omics data, (iii) cross-species gene mapping of complex traits via transcriptome-wide association studies, and (iv) high-quality results display and visualization. The PigBiobank will be updated timely with the development of the FarmGTEx-PigGTEx project, serving as an open-access and easy-to-use resource for genetically and biologically dissecting complex traits in pigs and translating the findings to other species.

Exploring the mechanism of artificial selection signature in Chinese indigenous pigs by leveraging multiple bioinformatics database tools.

BACKGROUND: Chinese indigenous pigs in Yunnan exhibit considerable phenotypic diversity, but their population structure and the biological interpretation of signatures of artificial selection require further investigation. To uncover population genetic diversity, migration events, and artificial selection signatures in Chinese domestic pigs, we sampled 111 Yunnan pigs from four breeds in Yunnan which is considered to be one of the centres of livestock domestication in China, and genotyped them using Illumina Porcine SNP60K BeadChip. We then leveraged multiple bioinformatics database tools to further investigate the signatures and associated complex traits. RESULTS: Population structure and migration analyses showed that Diannanxiaoer pigs had different genetic backgrounds from other Yunnan pigs, and Gaoligongshan may undergone the migration events from Baoshan and Saba pigs. Intriguingly, we identified a possible common target of sharing artificial selection on a 265.09 kb region on chromosome 5 in Yunnan indigenous pigs, and the genes on this region were associated with cardiovascular and immune systems. We also detected several candidate genes correlated with dietary adaptation, body size (e.g., PASCIN1, GRM4, ITPR2), and reproductive performance. In addition, the breed-sharing gene MMP16 was identified to be a human-mediated gene. Multiple lines of evidence at the mammalian genome, transcriptome, and phenome levels further supported the evidence for the causality between MMP16 variants and the metabolic diseases, brain development, and cartilage tissues in Chinese pigs. Our results suggested that the suppression of MMP16 would directly lead to inactivity and insensitivity of neuronal activity and skeletal development in Chinese indigenous pigs. CONCLUSION: In this study, the population genetic analyses and identification of artificial selection signatures of Yunnan indigenous pigs help to build an understanding of the effect of human-mediated selection mechanisms on phenotypic traits in Chinese indigenous pigs. Further studies are needed to fully characterize the process of human-mediated genes and biological mechanisms.

High Diversity of Epicoccum Associated with Leaf Spot on Italian Ryegrass in Southwestern China: Three New Species and Six New Records.

Italian ryegrass is widely cultivated for the productions of forage, hay and silage, due to its high nutritional value and good palatability. Leaf spots caused by fungi pose a serious threat to forage crops. In order to expand knowledge of fungi causing leaf spot in ryegrass (Lolium multiforum) in Sichuan, Yunnan, Chongqing and Guizhou of southwestern China, a comprehensive survey was undertaken from 2015 to 2022. Survey discovered that Epicoccum leaf spot (ELS) was a common and widespread disease, more serious at the late stage of growth (after late May); symptomatic leaf samples collected from the four different provinces were analyzed, and a total of 202 Epicoccum isolates were obtained; based on both multilocus phylogeny (ITS, LSU, TUB2, and RPB2) and morphology, 10 Epicoccum species were finally identified, including three novel species (E. endololii sp. nov., E. lolii sp. nov. and E. loliicola sp. nov.), six new host records (E. draconis, E. endophyticum, E. oryzae, E. plurivorum, E. thailandicum and E. tobaicum), and an unknown species (Epicoccum sp.1). Pathogenicity tests showed that E. endophyticum, E. endololii and Epicoccum sp.1 were non-pathogenic to Italian ryegrass, which were confirmed as endophytes in this study; other six species could infect Italian ryegrass and cause leaf lesions to different degrees, of which E. draconis was more aggressive (P ≤ 0.05). Coupling with the isolation rates and geographical distributions of these species, E. plurivorum was the predominant pathogen in Yunnan while E. oryzae and E. tobaicum in other three provinces. This work provides an initial understanding of the taxonomies, virulence and distributions of Epicoccum species associated with ELS of southwestern China, and lays a solid foundation for the diagnosis in the field, and scientific control of ELS on Italian ryegrass.

First Report of Pseudopithomyces palmicola Causing Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely cultivated for the production of forage, hay and silage worldwide, due to its high nutritional value and palatability (Feng et al. 2021). It has been infected by a number of foliar fungal diseases caused by different fungal pathogens (Xue et al. 2017, 2020; Victoria Arellano et al. 2021; Liu et al. 2023). In August 2021, three Pseudopithomyces isolates with similar colony characteristics were isolated from fresh samples of leaf spots on Italian ryegrass that was collected from the Forage Germplasm Nursery, Maming town of Qujing city, Yunnan province, China (25.53833°N, 103.60278°E). For specific isolation, tissue pieces (about 0.5 cm × 1 cm) from symptomatic leaves were surface-disinfested in 75% ethanol solution for 40s, rinsed three times in sterilized distilled water and air dried, plated on potato dextrose agar (PDA), and incubated at 25°C for 3 to 7 days in the dark. Following initial isolation, a representative isolate, KM42, was selected for further study. Colonies growing on PDA were cottony, white to grey, 53.8 to 56.9 mm diameter after 6 days in the dark at 25°C, with white and regular edge; in reverse, colonies were brown to dark brown in the center, and pale yellow toward the periphery with concentric rings. Under near-UV light at room temperature (20 ± 3°C), colonies were incubated on PDA for ten days to produce conidia. Conidia were globose or ellipsoid to amygdaloid, 1- to 3-transversely septate, 0- to 2-vertical septate, light brown to brown, measuring 11.6 to 24.4 × 7.7 to 16.8 µm (avg. 17.3 × 10.9 µm). The internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA(ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified with primers described by Chen et al. (2017). Sequences were deposited in GenBank (OQ875842 for ITS, OQ875844 for LSU, and OQ883943 for RPB2). BLAST analysis of all these three segments showed 100% (ITS: MF804527), 100% (LSU: KU554630), and 99.4% (RPB2: MH249030) identity with sequences of reported isolate CBS 143931 (= UC22) of Pseudopithomyces palmicola (Lorenzini et al. 2016; Liu et al. 2018). To fulfill Koch's postulates, four 12-week-old healthy Italian ryegrass plants were spray-inoculated separately with a mycelial suspension of about 5.4 × 102 CFU/ml of an isolate of P. palmicola. In addition, four control plants were sprayed with sterilized distilled water. All plants were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 22°C. Small brown to dark brown spots developed on leaves 10 days after inoculation; symptoms were not observed on control plants. Pathogenicity tests were conducted three times by the same method. The same fungus was re-isolated from the lesions, and confirmed by morphological and molecular methods as described above. To the best of our knowledge, this is the first report of P. palmicola causing leaf spot on Italian ryegrass in China or worldwide. This information will be useful to forage grass managers and plant pathologists in recognizing the disease and developing effective control measures.

Evaluation of Performance Tradeoffs When Using Mechanically Swept 1-D Linear Arrays for 3-D DCE-US.

Dynamic contrast-enhanced ultrasound imaging (DCE-US) may be used to characterize tumor vascular perfusion using metrics derived from time-amplitude curves (TACs). The 3-D DCE-US enables generation of 3-D parametric maps of TAC metrics that may inform on how perfusion varies across the entire tumor. The aim of this work was to understand the effect of low temporal sampling (i.e., < 1 Hz) typical of 3-D imaging using a swept 1-D array transducer on the evaluation of TAC metrics and the effect of transducer motion in combination with flow on 3-D parametric maps generated using both plane wave imaging (PWI) (seven angles) and focused imaging (FI). Correlation maps were introduced to evaluate the spatial blurring of TAC metrics. A research ultrasound scanner and a pulse-inversion algorithm were used to obtain DCE-US. The 2-D (frame rate 10 Hz) and 3-D (volume rate 0.4 Hz) images were acquired of a simple wall-less vessel phantom (flow phantom) and a cartridge phantom. Volumetric imaging provided similar TACs to that of the higher 2-D sampling rate. Varying sweep speed and acceleration/deceleration had little influence on the 3-D TAC compared to 2-D for both FI and PWI. Sweeping motion and limited temporal sampling (0.4 Hz) did not change the spatial correlation of TAC metrics measured using FI, whereas a small increase in correlation across the cartridge phantom was observed for PWI. This was attributed to grating lobe artifacts, broad beam spatial blurring, and incoherent compounding caused by motion. Increased correlation will reduce the spatial resolution with which inhomogeneity of vascular perfusion can be mapped supporting the choice of FI for DCE-US.

Evaluation of six machine learning classification algorithms in pig breed identification using SNPs array data.

Breed identification utilizing multiple information sources and methods is widely applicated in the field of animal genetics and breeding. Simultaneously, with the development of artificial intelligence, the integration of high-throughput genomic data and machine learning techniques is increasingly used for breed identification. In this context, we used 654 individuals from 15 pig breeds, evaluating the performance of machine learning and stacking ensemble learning classifiers, as well as the function of feature selection and anomaly detection in different scenarios. Our results showed that, when using a training set of 16 individuals per breed and 32 features (SNPs), the accuracy of breed identification with feature selection (eXtreme Gradient Boosting, XGBoost) could exceed 95.00% (nine breeds), and was improved by 7.04% over the results with random selection. For stacking ensemble learning, feature selection methods (including random selection method) were used before different base learners. When these base learners' training set had 16 individuals per breed and 32 features, the accuracy of stacking ensemble learning improved by 9.24% over the best base learner (nine breeds), but did not significantly increase the advantage over the models with XGBoost feature selection. When using a training set of 16 individuals and 512 features per breed, breed identification with anomaly detection (local outlier factor, LOF) and random selection could achieve an accuracy of 89.06% (15 breeds). These results show that machine learning could be an effective tool for breed identification and this study will also provide useful information for the application of machine learning in animal genetics and breeding.

Differences in the Characteristics and Pathogenicity of Pyrenophora Species Associated with Seeds of Italian Ryegrass.

Pyrenophora is a genus of pathogens that cause leaf damage and a common seedborne fungus of Italian ryegrass (Lolium multiflorum Lam.). To elucidate the main seedborne Pyrenophora species, 36 seed samples of Italian ryegrass were collected; in total, 113 strains, representing 12.3% of all isolated strains (921), were identified as Pyrenophora species using the identity of ITS sequences in NCBI and the similarity of morphological characteristics. P. dictyoides (97) was the most frequent species. By pure culture technique, 24 representative pure isolates were obtained for further study. Based on DNA analysis of multiple loci (ITS, LSU, GPDH, CHS-1, and RPB1) and morphological characters, eight Pyrenophora species were identified, P. avenicola, P. chaetomioides, P. dictyoides, P. lolii, P. nobleae, P. teres, P. triseptata, and P. tritici-repentis; among them, P. avenicola, P. tritici-repentis, and P. triseptata were newly reported on Italian ryegrass worldwide. Seed inoculation showed that P. dictyoides, P. lolii, and P. teres remarkably decreased the final germination percentages and germination indexes compared with control treatments (P ≤ 0.05); and plant inoculation showed that P. dictyoides, P. lolii, and P. nobleae could cause typical brown spot in vivo with a higher infection rate (P ≤ 0.05). In conclusion, pathogenicity tests showed that all Pyrenophora species could both inhibit seed germination and infect Italian ryegrass to different degrees; among them, P. dictyoides was the most important seedborne pathogen based on the combination of its isolation and infection rate, followed by P. lolii and P. nobleae. The data generated in this study are helpful for the accurate identification of Pyrenophora species and the development of seedborne disease management strategies.

Meta-analysis of genome-wide association studies uncovers shared candidate genes across breeds for pig fatness trait.

BACKGROUND: Average backfat thickness (BFT) is a critical complex trait in pig and an important indicator for fat deposition and lean rate. Usually, genome-wide association study (GWAS) was used to discover quantitative trait loci (QTLs) of BFT in a single population. However, the power of GWAS is limited by sample size in a single population. Alternatively, meta-analysis of GWAS (metaGWAS) is an attractive method to increase the statistical power by integrating data from multiple breeds and populations. The aim of this study is to identify shared genetic characterization of BFT across breeds in pigs via metaGWAS.  RESULTS: In this study, we performed metaGWAS on BFT using 15,353 pigs (5,143 Duroc, 7,275 Yorkshire, and 2,935 Landrace) from 19 populations. We detected 40 genome-wide significant SNPs (Bonferroni corrected P < 0.05) and defined five breed-shared QTLs in across-breed metaGWAS. Markers within the five QTL regions explained 7 ~ 9% additive genetic variance and showed strong heritability enrichment. Furthermore, by integrating information from multiple bioinformatics databases, we annotated 46 candidate genes located in the five QTLs. Among them, three important (MC4R, PPARD, and SLC27A1) and seven suggestive candidate genes (PHLPP1, NUDT3, ILRUN, RELCH, KCNQ5, ITPR3, and U3) were identified. CONCLUSION: QTLs and candidate genes underlying BFT across breeds were identified via metaGWAS from multiple populations. Our findings contribute to the understanding of the genetic architecture of BFT and the regulating mechanism underlying fat deposition in pigs.

Insights into the architecture of human-induced polygenic selection in Duroc pigs.

BACKGROUND: As one of the most utilized commercial composite boar lines, Duroc pigs have been introduced to China and undergone strongly human-induced selection over the past decades. However, the efficiencies and limitations of previous breeding of Chinese Duroc pigs are largely understudied. The objective of this study was to uncover directional polygenic selection in the Duroc pig genome, and investigate points overlooked in the past breeding process. RESULTS: Here, we utilized the Generation Proxy Selection Mapping (GPSM) on a dataset of 1067 Duroc pigs with 8,766,074 imputed SNPs. GPSM detected a total of 5649 putative SNPs actively under selection in the Chinese Duroc pig population, and the potential functions of the selection regions were mainly related to production, meat and carcass traits. Meanwhile, we observed that the allele frequency of variants related to teat number (NT) relevant traits was also changed, which might be influenced by genes that had pleiotropic effects. First, we identified the direction of selection on NT traits by [Formula: see text], and further pinpointed large-effect genomic regions associated with NT relevant traits by selection signature and GWAS. Combining results of NT relevant traits-specific selection signatures and GWAS, we found three common genome regions, which were overlapped with QTLs related to production, meat and carcass traits besides "teat number" QTLs. This implied that there were some pleiotropic variants underlying NT and economic traits. We further found that rs346331089 has pleiotropic effects on NT and economic traits, e.g., litter size at weaning (LSW), litter weight at weaning (LWW), days to 100 kg (D100), backfat thickness at 100 kg (B100), and loin muscle area at 100 kg (L100) traits. CONCLUSIONS: The selected loci that we identified across methods displayed the past breeding process of Chinese Duroc pigs, and our findings could be used to inform future breeding decision.

A Nude Mouse Model of Orthotopic Liver Transplantation of Human Hepatocellular Carcinoma HCCLM3 Cell Xenografts and the Use of Imaging to Evaluate Tumor Progression.

BACKGROUND This study aimed to develop a nude mouse model of orthotopic liver transplantation of HCCLM3 human hepatocellular carcinoma (HCC) cell xenografts and the use of imaging and histology to evaluate tumor development and progression. MATERIAL AND METHODS HCCLM3 cells were injected subcutaneously into 25 healthy male athymic BALB/c (nu/nu) nude mice. The tumors that developed were transplanted into the liver of a new set of nude mice. After four weeks and six weeks, the mice were imaged using ultrasound (US), software-assisted contrast-enhanced ultrasound (CEUS), fluorodeoxyglucose-positron emission tomography (FDG-PET). Histology was performed on the liver and liver tumors, and included immunohistochemistry for vascular endothelial growth factor (VEGF), CD31, CD34, and alpha-smooth muscle actin (alpha-SMA). RESULTS The success rate for orthotopic tumor transplantation in the mouse liver was 90% (18/20). Liver tumors measured 11.8±2.6 mm in diameter and 525.9±250.8 mm3 in volume on the sixth week. CEUS showed rapid wash-in and washout in the liver tumors, and PET showed low tumor cell metabolism. Bone metastases were present in 45% (9/20) of mice in the sixth week. Immunohistochemistry showed positive expression for VEGF, CD31, CD34, and alpha-SMA. CONCLUSIONS The nude mouse orthotopic liver transplantation model of human HCC was shown to be a reliable model that has the potential for future research on the pathogenesis and progression of HCC and studies on drug development.

Genetic Diversity of Indigenous Pigs from South China Area Revealed by SNP Array.

To investigate the genetic diversity, population structure, extent of linkage disequilibrium (LD), effective population size (Ne), and selection signatures in indigenous pigs from Guangdong and Guangxi in China, 226 pigs belonging to ten diverse populations were genotyped using single nucleotide polymorphism (SNP) chips. The genetic divergence between Chinese and Western pigs was determined based on the SNP chip data. Low genetic diversity of Dahuabai (DHB), Luchuan (LC), Lantang (LT), and Meihua (MH) pigs, and introgression of Western pigs into Longlin (LL), MH, and Yuedonghei (YDH) pigs were detected. Analysis of the extent of LD showed that indigenous pigs had low LD when pairwise SNP distance was short and high LD when pairwise SNP distance was long. Effective population size analysis showed a rapid decrease for Chinese indigenous pigs, and some pig populations had a relatively small Ne. This result indicated the loss of genetic diversity in indigenous pigs, and introgression from Western commercial pigs. Selection signatures detected in this study overlapped with meat quality traits, such as drip loss, intramuscular fat content, meat color b*, and average backfat thickness. Our study deepened understanding of the conservation status and domestication of Chinese indigenous pigs.

Effect of dry heat treatment with xanthan on waxy rice starch.

Waxy rice starch was impregnated with xanthan and heat-treated in a dry state. The effects on the pasting and rheological properties of the treated starch-xanthan mixture were evaluated. Swelling of the granule was restricted, and a continuous rise of the viscosity during pasting was provided for the treated sample. After pasting, the gel forming ability of the treated starch was strengthened, as both storage and loss modulus increased and tan δ decreased. The paste also owned the highest zero order Newtonian viscosity and yield stress. An increase in starch particle size of the dry heated starch-xanthan mixture suggested a cross linking of the starch granules by the xanthan polymers. An increase of crystallinity was observed for the starch after dry heat treatment, but with the addition of xanthan the amorphous region of the granule became more resistant to dry-heating. The melting enthalpy was found to be correlated with the crystallinity.

Effect of polysaccharides on the gelatinization properties of cornstarch dispersions.

Konjac glucomannan (KG, neutral), carboxymethylcellulose (CMC, negatively charged), and chitosan (positively charged) were added to cornstarch dispersions to study the effect of polysaccharide-starch interactions on starch gelatinization properties. Pasting and retrogradation properties were measured with a rheometer and DSC. Swelling properties of the starch granules were determined by solubility index, swelling power, and particle size distribution. Depending on the nature of the different polysaccharides, viscosities of cornstarch dispersions were affected differently. The particle size distributions were not influenced by the addition of any of the polysaccharides. Swelling results showed that the KG and CMC molecules interacted with the released or partly released amylose in the cornstarch dispersions. This was correlated with the short-term retrogradation of the starch pastes being retarded by the additions of KG and CMC. However, the chitosan molecules appeared not to associate with the amylose, so the retrogradation of the chitosan-cornstarch dispersions was not retarded.