Submit or Track your Manuscript LOG-IN

Whole-genome Sequencing Reveals the Genetic Relationships and Selection Signatures of the Min Pig

PJZ_54_3_1187-1198

Whole-genome Sequencing Reveals the Genetic Relationships and Selection Signatures of the Min Pig

Zhang Dong-jie1, He Xin-miao1,2, Wang Wen-tao1,2 and Liu Di1,2*

1Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, P.R. China

2Key Laboratory of Combining Farming and Animal Husbandry, Ministry of Agriculture, Harbin 150086, P.R. China

ABSTRACT

The Min pig is indigenous to China. The genetic background of this breed was previously unclear, limiting the utility of the Min pig. In this study, the whole genomes of ten Min pigs and four Northeast wild boars were sequenced and the analysis yielded 8,988,338 non-redundant SNPs plus 1,231,680 InDels. A phylogenetic tree was constructed and a principal component analysis (PCA) was performed based on previously published SNP data from 66 individual pigs. Both analyses indicated the Min pig fell between the European and Asian pigs, while the Northeast wild boar was closely related to the Asian domestic and wild pig breeds. Selective sweep analysis indicated that 181 genes in the Min pig genome had been subjected to selection, including several genes encoding zinc finger proteins. Additional genes associated with myokinesis and lipid metabolism were also identified as under selection. Only SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) interactions in the vesicular transport pathway were identified as under selection (P=0.0029). This study describes the genomic framework of the Min pig and identifies signatures of selection. These results provide a useful genomic background for further studies of the genetic mechanisms associated with important economic traits in the Min pig.


Article Information

Received 09 July 2020

Revised 28 September 2020

Accepted 08 October 2020

Available online 20 May 2021

(early access)

Published 19 February 2022

Authors’ Contribution

LD designed the experiment. ZD-J and HX-M performed the experiment. WW-T processed the data. All authors wrote the manuscript.

Key words

Min pig, Northeast wild boar, Whole genome sequencing, Genetic relationship, Selection signatures

DOI: https://dx.doi.org/10.17582/journal.pjz/20200709040722

* Corresponding author: liudi1963@163.com

0030-9923/2022/0003-1187 $ 9.00/0

Copyright 2022 Zoological Society of Pakistan



INTRODUCTION

Pigs are one of the most important human-domesticated animals worldwide. Globally, pork accounts for a high proportion of human meat consumption, especially in China. In 2018, 62% of all meat consumed in China was pork. Pigs are also useful animal models for biomedical research (Yang et al., 2015). Pigs have been domesticated for thousands of years, and were originally domesticated in multiple regions (Giuffra et al., 2000; Bosse et al., 2014). Thus, domestic pigs represent a rich genetic resource.

China is one of the original regions of pig domestication. Wild boars began to be domesticated in China about 10,000 years ago (Xiang et al., 2017). At present, there are 76 indigenous pig breeds in China. Comparing commercial pigs, the indigenous Chinese breeds are highly fertile, have high disease resistance, tolerate coarse feed, have a slow growth rate, and have a low percentage of lean meat (China National Commission of Animal Genetics Resources, 2011). However, Chinese pig breeds have been subjected to almost no high-intensity artificial breeding programs. As the phenotypic and genetic diversities of Chinese pigs are richer than those of commercial pig breeds, the Chinese pigs represent ideal models for the improvement of economic traits and genetic research (Guo et al., 2015; Kirthika et al., 2017).

The Min pig is one of the oldest and most famous indigenous pig breeds in China. Although this breed is mainly distributed in the northeastern China, it was introduced into the United States in the last century (Young, 1992). The genetic diversity of the Min pig is very rich. According to historical records, the Min pig migrated from Shandong province to northeastern China with humans, about 400 years ago. However, pigs in northeastern China were independently domesticated 8,000–3,500 BP (before present) (Xiang et al., 2017). Therefore, the Min pig may have been crossed with native pig breeds. A recent study identified a long fragment (~48Mb) in the X chromosome of the Min pig that was derived from ancient interspecies introgression. At the same time, the Min pig has two haplotypes: The European and the Northern Chinese (Ai et al., 2015). These results illustrate the complexity of the genetic background of the Min pig. Understanding the genetics of this breed is a prerequisite for making full use of its germplasm. The development of next-generation sequencing technology has made sequencing the complete genome of a species easy and cheap. Next-generation sequencing is a fast and accurate way to discover the hereditary basis of important breed characteristics.

In this study, the whole genomes of ten Min pigs and four Northeast wild boars were sequenced, and the genomic characteristics of these pigs were analyzed. The SNPs data of Northeast wild boar, Min pig and Yorkshire were compared, because their genomic differences represented the various degrees of artificial selection. The evolutionary identity of the Min pig was investigated by comparing Min pig genomes with the previously published genomes of other pig breeds, including various Chinese indigenous pigs, European pig breeds, and some wild boars. Finally, artificially or naturally selected genes were identified.

MATERIALS AND METHODS

Ethical aspects

The experimental protocol used in this study was approved by the Heilongjiang Academy of Agricultural Sciences, Harbin, China (2015-5).

Animals and sampling

Ear tissue samples were collected from ten Min pigs at the scientific research base of the Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences (Harbin, China). No lineage relationships existed among any of the sampled pigs within three generations. Ear tissue samples were also collected from four Northeast wild boars in the Xiao Xing’an Ling forest zone of Heilongjiang province, China.

Genomic sequencing strategy

DNA was extracted from each sample of ear tissue. Total DNA was isolated using the QIAamp DNA Mini Kit (Qiagen 51304) following the manufacturer’s instructions. The genomes were sequenced using an Illumina (San Diego, CA, USA) Hiseq2000 with PE500 libraries, in paired-end 2×100 mode. The raw reads obtained after library construction and sequencing were first cleaned by removing the 3’-ends using cut adapt 1.2.1, allowing a 10% base error rate and requiring ≥10 bp overlap (AGATCGGAAG). Next, sequences were removed if they had an average quality score ≤ Q20 within a 5-bp sliding window or ambiguous (“N”) bases, or if they were shorter than 50 bp.

High-quality paired-end reads were mapped to the pig reference genome sequence (Sus scrofa 10.2) using Burrows-Wheeler Aligner (BWA 0.7.12). The reference sequence was indexed and a suffix array (SA) coordinate alignment was generated for each read using the “aln–k2–l32–i5-o1–q0” command. The SA coordinates were converted to chromosomal coordinates and output in sequence alignment map (SAM) format with command “sampe–a2000.” The alignments were improved using Picard (Picard tools 1.119) (http://sourceforge.net/projects/picard/): The “Fix Mate Information” command was used to ensure that all mate-pair information was synchronized between each read and its mate pair, and the “Mark Duplicates” command was used to mark potential polymerase chain reaction (PCR) duplicates. Where multiple read pairs had identical external coordinates, only the pair with the best mapping quality was retained, and the others were marked as PCR duplicates. A local realignment of the mapped reads around InDels was performed using GATK (GATK3.0): First, the “Realigner Target Creator” command was used to identify suspicious intervals that were likely in need of realignment, and the “IndelRealigner” command was then used to realign the identified intervals.

SNP and InDel calling from sequencing data

After alignment, SNP calling for the two populations (Min pigs and Northeast wild boars) was performed using the Bayesian approach as implemented in the GATK. GATK uses a Bayesian genotype likelihood model to estimate the most likely genotypes and allele frequencies in a given population simultaneously, and generates an accurate posterior probability of a segregating variant allele at each locus, as well as an accurate posterior probability of each genotype in each sample. The “UnifiedGenotyper” command with parameters “-stand_call_conf 50-stand_emit_conf 30.0” was used to identify SNPs. SNPs were filtered, and only high-quality SNPs were retained. High-quality SNPs were defined as those which met several criteria: coverage depth ≥8 and ≤1000; root mean square (RMS) mapping quality ≥40; variant confidence/quality by depth ≥2; Phred-scaled P-value, using Fisher’s exact test to detect strand bias ≤60; consistency of the site with at most two segregating haplotypes ≤13.0; Z-score from the Wilcoxon rank sum test of Alt vs. Ref read mapping qualities ≥-12.5; Z-score from the Wilcoxon rank sum test of Alt vs. Ref read position bias ≥-8.0; and the missing percentage of samples within each group ≤50%.

InDel calling was performed using the “UnifiedGenotyper” command with the parameters “-stand_call_conf50-stand_emit_conf30.0” in GATK. Only high-quality InDels were retained, where high-quality InDels were defined as those which met four criteria: coverage depth ≥8 and ≤1000; Phred-scaled P-value, using Fisher’s exact test to detect strand bias ≤200; variant confidence/quality by depth ≥2; and Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias ≥-20.0.

Genomic population sequences

To explore the genetic relationships among worldwide pig populations, genome sequence data for 66 individual pigs (including indigenous pigs and wild boars) were downloaded from the NCBI Genbank and these sequences were analyzed as described above. A phylogenetic tree was constructed using MEGA5 with the novel sequencing data and previously published data and the application of the neighbor-joining method. Tree reliability was assessed using 1000 bootstrap replicates. The numbers presented for each clade represent bootstrap support values given as percentages.

A principal component analysis (PCA) of the autosomal SNPs was performed using EIGENSOFT 5.0.2. Eigen vectors were obtained from the covariance matrix using reigen in R, and the significance of differences among vectors was determined using the Tracy-Widom test. SNPs were compared among the Min pig, the Northeast wild boar, and the pig dbSNP138 data (NCBI: txid9823). SNPs from the Min pig, Yorkshire, and Northeast wild boar populations were called and filtered as described above, except that coverage depth was set to ≥8 and ≤1000 for Min pigs, ≥8 and ≤500 for Yorkshire boars, and ≥8 and ≤400 for Northeast wild boars.

Selective sweep analysis

A sliding-window approach (500-kb windows sliding in 50-kb steps) was used to calculate polymorphism levels (using θπ pairwise nucleotide variation as a measure of variability), genetic differentiation (Fst), and selection statistics (Tajima’s D, a measure of selection in the genome) between the Min pig and the Northeast wild boar. The distributions of the θπ ratios (θπ, Min/θπ, northeast) and the Fst values were used to identify regions with significant signatures of the selective sweeps. An empirical procedure was used to select windows in the empirical distribution with significant low and high θπ ratios (5% left and right tails, where the θπ ratios were 0.728 and 1.367, respectively) in conjunction with significantly high Fst values (5% right tail, where Fst was 0.361). These regions of the genome were considered to have strong signals of selective sweep, and might harbor genes that have undergone selection.

Functional enrichment of selected genes

Functional enrichments in Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and InterPro domains were explored using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Genes showing evidence of selection were mapped to their respective human orthologs, and the lists were submitted to DAVID to test the enrichment of various biological categories, including GO biological processes (GO-BP), GO molecular functions (GO-MF), KEGG pathways, and InterPro domains. For each test, all known genes were used as the background, and the Benjamini-corrected modified Fisher Exact P values (EASE score) were calculated to determine the significance of the enrichment. Only categories with a P-value <0.05 were considered to be significantly enriched.

RESULTS

Genomic sequencing information

Based on the size of the Sus scrofa genome (2.8 Gb), the genomic sequencing coverage was about 7.8× for the Min pigs and 9× for the Northeast wild boars. The analysis identified 8,988,338 non-redundant SNPs plus 1,231,680 InDels from the Min pigs, and 11,345,128 non-redundant SNPs plus 1,121,653 InDels from the Northeast wild boars (Fig. 1A).

In both the domesticated Min pigs and the Northeast wild boars, many SNPs were intergenic and intronic; only a few SNPs were identified in the exonic regions or the untranslated regions (UTRs) (Table I). In the Min pigs, 6,199,989 heterozygous SNPs (68.98% of 8,988,338) and 2,788,349 homozygous SNPs (31.02% of 8,988,338) were identified. In the Northeast wild boars, 6,166,077 heterozygous SNPs (54.35% of 11,345,128) and 5,179,051 homozygous SNPs (45.65% of 11,345,128) were identified. The number of SNPs distributed on each chromosome was positively correlated with chromosome length (Fig. 1C).

 

Table I. Distribution of SNPs in Min pigs and Northeast wild boars.

Category

Min pig

Northeast wild boar

Exonic

43,108

51,017

nc_transcript

42,276

46,904

Intronic

1,991,046

2,499,832

UTR

31,872

38,293

Splicing

261

308

Upstream/Downstream

574,446

702,931

Intergenic

5,572,866

6,935,773

 

Exonic, A transcript variant occurring within an exon; nc_transcript, A transcript variant of a non coding RNA; Intronic, A transcript variant occurring within an intron; UTR, A UTR variant of the 5’ UTR or 3’ UTR; Splicing, A splice variant that changes the 2 base region at the 5’ end of an intron; Upstream/downstream, A sequence variant overlaps with the 1 kb region downstream or upstream of a gene; Intergenic, A sequence variant located in the intergenic region, between genes.

 

 

SNPs from Min pigs and Northeast wild boars were called and filtered as described above, by setting coverage depth ≥8 and ≤1400. This yielded 27,208,632 SNPs across all fourteen individual genomes, of which 18,190,197 (66.85%) were intergenic, 6,722,554 (24.71%) were intronic, and 143,272 (0.53%) were exonic (Fig. 1B). The identified SNPs were compared to those in the pig dbSNP 138 database, which showed that 68.06% of the SNP variants in pig dbSNP (19,469,459 SNPs) were in the SNP dataset generated in this study, but 7,739,173 of the SNP variants identified in this study were absent from the dbSNP 138 database (Fig. 1B). These novel SNPs increased the number of known porcine genetic variants.

Yorkshire pigs are a mature cultivated pig breed. SNPs from Min pigs, Yorkshire pigs (ERS177318, ERS177319, ERS177320, ERS177322, ERS177325), and Northeast wild boars were called and filtered as described above, with coverage depths set to ≥8 and ≤1000 for Min pigs; to ≥8 and ≤500 for Yorkshire pigs; and to ≥8 and ≤400 for Northeast wild boars. This analysis identified 22,368,881 SNPs across the ten Min pigs; 17,585,639 SNPs across the four Northeast wild boars; and 9,854,629 SNPs across the five Yorkshire pigs. Of the identified SNPs, 5,398,231 were shared among all three breeds, while 6,858,528 SNPs were unique to the Min pigs; 4,642,136 SNPs were unique to the Northeast wild boars; and 1,092,046 SNPs were unique to the Yorkshire pigs.

Phylogenetic analysis and demographic distribution

This study identified 28,411,844 SNPs in the 66 previously published pig genomes. A phylogenetic tree was constructed based on the novel sequencing data generated here and the previously published data (Fig. 2), which shows that the Min pig clustered together with the Northeast wild boars and several European pig breeds (e.g., Duroc, Yorkshire, and Landraces). The constructed phylogeny was then analyzed with the PCA (Fig. 3). In the PCA, the first eigenvector geographically distinguished 36 Asian individuals from 26 European individuals and one African individual; while the second eigenvector captured the biological differences between the pigs. Notably, a previous study also showed that Min pigs have a European haplotype in their X-linked selective sweep region, and this region has an exceptionally low recombination rate (Ai et al., 2015).

 

Screening of selected genes

The results of these analyses suggested that the Min pig had slightly lower levels of polymorphism as compared to the Northeast wild boar (median θπ; Min/θπ; Northeast=0.9855) (Fig. 4A). Genomic regions with strong signals of selective sweep in the Min pig spanned 15.71 Mb (0.559% of the genome, containing 181 genes). In Northeast wild boar, the regions with strong signals of selective sweep spanned 29.81 Mb (1.061% of the genome, containing 411 genes) (Fig. 4B). Of the 181 genes showing evidence of selection in the Min pig, 118 have been annotated (Table II). The proteins encoded by these 118 candidate genes included several zinc finger proteins: ZNF259 (zinc finger protein 259), ZNF365 (zinc finger protein 365), ZNF384 (zinc finger protein 384), ZNF410 (zinc finger protein 410), ZNF646 (zinc finger protein 646), ZNF668 (zinc finger protein 668), ZNF713 (zinc finger protein 713), ZCCHC9 (zinc finger, CCHC domain containing 9), and ZBTB11 (zinc finger and BTB domain containing 11). Signatures of selection were also identified in some genes associated with myokinesis and lipid metabolism, including CKMT2 (creatine kinase mitochondrial 2), MYO1C (myosin 1C), LCN9 (lipocalin 9), LCN15 (lipocalin 15), APOA5 (apolipoproteinA-V), and CRHR2 (corticotropin- releasing hormone receptor 2). In addition to these, other genes identified as having been under selection were VRTN (vertebrae development homolog), which is related to the number of vertebrae (Ren et al., 2012), and OPA1 (opticatrophy 1), which is associated with autosomal dominant optic atrophy (Jin et al., 2015). In

 

addition, OR2B6 (olfactory receptor, family 2, subfamily B, member 6) and ARHGAP 32 (Rho GTPase activating protein 32) overlapped with the top signals of selective sweep.

Of the 411 genes showing evidence of selection in the Northeast wild boar, 279 were annotated. These genes were mainly related to natural selection. Some of them are associated with the nervous system, including olfactory receptors (e.g., OR2B6, OR8b8, and OR8b4) (Monahan et al., 2015), PDCL3 (Srinivasan et al., 2015), and TAC3 (Shankar et al., 2015); some are related to immunity, including NLR family genes, pyrin domain containing genes (e.g.,NLRP4 and NLRP11) (Eibl et al., 2012; Tadaki et al., 2011), IFN-OMEGA-2 (Zhao et al., 2009), Foxn1 (Ruan et al., 2014), and OSCAR (Barrow et al., 2015); some are related to male reproduction, including HSD17B6 (Ishizaki et al., 2013), ACRBP (Vilagran et al., 2013), ELSPBP1, and PRM1 (D’Amours et al., 2012; Dogan et al., 2015); and some are related to mitochondrial energy metabolism, including COX6A1 and NDUFA9. Of all the genes showing evidence of selection, only Arhgap32 was identified in both the Min pig and the Northeast wild boar.

Functional enrichment of selected genes

The enrichment of genes showing evidence of selection in Min pigs and Northeast wild boars was explored with DAVID. Some pathways, GO terms, and Interpro domains were significantly enriched (P<0.05). In the Min pig, only soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) interactions

 

Table II. Key selected genes in Min pigs.

Gene

Fst

Theta_Pi

Description

Pathways from biosystems

SPTSSB

0.41

0.37

Serine palmitoyltransferase, small subunit B

Metabolism of lipids and lipoproteins

LDHB

0.31

0.61

L-Lactate dehydrogenase

Glucagon signaling pathway

HIBCH

0.31

0.43

3-Hydroxyisobutyryl-CoA hydrolase

Amino Acid metabolism, Carbon metabolism

PSPH

0.29

0.62

Phosphoserine phosphatase

Amino acid synthesis and interconversion (transamination)

CAPZA2

0.29

0.35

Capping protein (actin filament) muscle Z-line, alpha 2

Advanced glycosylation endproduct receptor signaling

ZCCHC9

0.28

0.58

Zinc finger, CCHC domain containing 9

Unknown

PLCH1

0.28

0.58

Phospholipase C, eta 1

D-myo-inositol (1,4,5)-trisphosphate biosynthesis

CKMT2

0.28

0.61

Creatine kinase, mitochondrial 2 (sarcomeric)

Arginine and proline metabolism; Creatine metabolism

PISD

0.28

0.62

Phosphatidylserine decarboxylase

FOXA1 transcription factor network; Glycerophospholipid biosynthesis

ZNF384

0.26

0.56

Zinc finger protein 384

Unknown

ZNF713

0.26

0.51

Zinc finger protein 713

Gene Expression

MYO1C

0.26

0.55

Myosin IC

Insulin Signaling

CERS6

0.25

0.58

Ceramide synthase 6

Ceramide biosynthesis

SEMA3C

0.24

0.34

Semaphorin 3C

Axon guidance

ZNF410

0.23

0.33

Zinc finger protein 410

Unknown

COQ6

0.23

0.29

Coenzyme Q6 monooxygenase

Metabolism of lipids and lipoproteins; Ubiquinol biosynthesis

LCN15

0.23

0.48

Lipocalin 15

Transmembrane transport of small molecules

VRTN

0.21

0.32

Vertebrae development homolog

Unknown

LCN9

0.21

0.62

Lipocalin 9

Transmembrane transport of small molecules

STRBP

0.21

0.36

Spermatid perinuclear RNA binding protein

Unknown

OPA1

0.20

0.63

OPA1 mitochondrial dynamin like GTPase

Regulation of Apoptosis

APOA5

0.20

0.60

Apolipoprotein A5

Chylomicron-mediated lipid transport

CRHR2

0.20

0.53

Corticotropin releasing hormone receptor 2

Corticotropin-releasing hormone signaling pathway

 

in the vesicular transport pathway were identified (P=0.0029); and the VAMP1, STX4, STX6, and STX1B genes were involved. Several GO terms and Interpro domains were enriched, including SNAP receptor activity (enriched with STX4, STX6, and STX1B), phospho lipid binding (enriched with PIK3C2G, ZFYVE28, APOA5, TRIM72, ARHGAP32, and SNX19), thiolester hydrolase activity (enriched with PSMD14, USP13, ACOT12, and HIBCH), and progesterone metabolic processes (enriched with AFP and DHRS9). In the Northeast wild boar, only the ABC transporter pathway was identified (P=0.0036). However, many genes were identified in the GO and Interpro analyses (Table III).

DISCUSSION

Many diverse pig breeds are indigenous to China. The appearances, body sizes, and productive capacities of these breeds differ significantly due to contrasting geographical environments, customs, and ethnicities. Pigs have been an important human food resource for many thousands of years. However, due to slow growth and a low percentage of lean meat, many indigenous pig breeds have been replaced by introduced pig breeds, including the Landrace, Duroc, and Yorkshire. Some Chinese pig breeds have become useful resources for academic research, including the Wuzhishan pig (Xu et al., 2019), Laiwu pig (Liu et al., 2019), Tibetan pig (Zhang et al., 2019), Taihu pig (Wang et al., 2019), and the Min pig (Liu et al., 2017).

In this study, the complete genomes of ten Min pigs and four Northeast wild boars were sequenced. Northeast wild boars have inhabited the same localities as Min pigs for hundreds of years. Here, 6,858,528 SNPs unique to the Min pig were identified, as were 4,642,136 SNPs unique to

 

Table III. GO, pathway and interpro analysis results.

Category

Term

P-Value

Genes cluster in this term

Min pig

GOTERM_MF_FAT

SNAP receptor activity

0.0114

STX4, STX6, STX1B

GOTERM_MF_FAT

Phospholipid binding

0.0123

PIK3C2G, ZFYVE28, APOA5, TRIM72, ARHGAP32, SNX19

GOTERM_MF_FAT

Thiolester hydrolase activity

0.0451

PSMD14, USP13, ACOT12, HIBCH

GOTERM_BP_FAT

Progesterone metabolic process

0.0465

AFP, DHRS9

KEGG_PATHWAY

SNARE interactions in vesicular transport

0.0029

VAMP1, STX4, STX6, STX1B

INTERPRO

Target SNARE coiled-coil region

0.0158

STX4, STX6, STX1B

INTERPRO

Potassium channel

0.0213

KCNAB1, KCNAB3

INTERPRO

HAD-superfamily hydrolase

0.0213

PSPH, PHOSPHO2

Northeast wild boar

GOTERM_MF_FAT

ATPase activity, coupled to transmembrane movement of substances

0.0073

ABCA3, ABCB1, ABCC5, ATP5F1B, ABCB4, ABCC1, ATP6V0C

GOTERM_BP_FAT

Negative regulation of protein kinase activity

0.0129

PDCD4, PDPK1, CBLC, PAK2, TSC2, NF2

GOTERM_BP_FAT

Negative regulation of protein kinase cascade

0.0197

SOCS1, TSC2, NF2, IL1RL1

GOTERM_BP_FAT

Cell-substrate adhesion

0.0207

TNFRSF12A, BCAM, PDPK1, ANTXR1, PKD1, ECM2

GOTERM_BP_FAT

Response to endogenous stimulus

0.0304

TXN2, SLC18A2, CAV2, SI, ADCY9, PDPK1, SOCS1, ABCC5, MGP, MSI1, EIF2B5, PLA2G1B, ABCB4

GOTERM_BP_FAT

Regulation of cell-substrate adhesion

0.0373

TSC2, COL8A1, NF2, ECM2

GOTERM_BP_FAT

Ion transport

0.0401

SLC40A1, CAV2, GABRD, KCNV1, SCN3B, KCTD5, CNNM3, PKD1, TRPM4, STEAP1, KCNK1, CNNM4, HTR3C, SLC12A6, NMUR2, SLC8A2, ATP5F1B, SLC12A7, SLC17A7, ATP6V0C

GOTERM_BP_FAT

DNA integration

0.0403

HMGA1, PPFIBP1, XRCC4

GOTERM_BP_FAT

Neurotransmitter transport

0.0444

SLC18A2, PCLO, PPFIA3, SLC6A20, SLC17A7

GOTERM_BP_FAT

Regulation of phosphorus metabolic process

0.0490

PDCD4, PKMYT1, SOCS1, CBLC, BMPR1A, NF2, AXIN1, PLA2G1B, ADCY9, SMG6, PDPK1, MAP3K21, TSC2, PAK2

KEGG_PATHWAY

ABC transporters

0.0036

ABCA3, ABCB1, ABCC5, ABCB4, ABCC1

INTERPRO

ABC transporter

0.0021

ABCA3, ABCB1, SMC3, ABCC5, ABCB4, ABCC1

 

the Northeast wild boar. More SNPs were identified in the Northeast wild boar than in the Min pig, and more SNPs were identified in the Min pig than in the Yorkshire pig. This pattern was consistent with the degrees of artificial selection imposed upon each breed.

The phylogeny developed here suggested that European pig breeds (including wild boars and domestic pigs) are distinct from Asian pig breeds (including wild boars and domestic pigs), consistent with previous studies (Choi et al., 2014). We were surprised that Min pig fell between the European and Asian pig breeds, and did not form a cluster with the Asian pig breeds like the Northeast wild boar. We therefore speculate that Min pig might have hybridized with European pig breeds at some point since the earliest domestication of this breed. Currently, the Min pig is primarily bred in Heilongjiang province, in the northernmost part of China. Only the Heilongjiang River separates Heilongjiang province from Russia, and cross-border trade has been common for hundreds of years. In addition, the Heilongjiang River is frozen for almost four months of the year, providing convenient conditions for gene flow among different pig breeds. Previous studies of the 60K SNP genotype data indicated that pig breeds indigenous to Russia, Belorussia, Kazakhstan, and Ukraine clustered with European pig breeds and that the Min pig clustered with Chinese breeds, including the Luchuan, Erhualian, Laiwu, Hetao large ear, and Tibetan (Traspov et al., 2016). However, in the neighbor-joining tree produced here, the Min pig is the Chinese pig breed that is closest to the European pig breeds.

Next-generation sequencing technology allows a clearer understanding of the origin and evolution of a species (Groenen et al., 2012; Li et al., 2013). The current study found little genetic overlap among the genomes of different pig breeds. It is possible that many genomic differences exist among breeds due to different breeding goals and living environments (Choi et al., 2015; Ma et al., 2015). These results also highlight the molecular mechanisms that potentially underly the various pig phenotypes. For example, the Yorkshire and Landrace breeds have both been subjected to intensive selection for fast growth, a high percentage of lean meat, and large litters. Therefore, the identification of the genes GHR, IGF1R, and IGF2R as showing evidence of genetic selection was not surprising (Wang et al., 2018). In addition, the genes of Tibetan wild boars, which live in high altitudes, show evidence of selection on genes associated with hypoxia, olfaction, energy metabolism, and the drug response (Li et al., 2013).

Min pig has a very special trait, which is cold tolerance. It lives in high-latitudes (47–53° N, 121–141° E), in regions that are very cold, with long winters. Min pigs are well adapted to temperatures that range from 39°C in summer to -25°C in the winter. Thus, that the identification of genes associated with myokinesis and lipid metabolism as being under selection was not surprising. Indeed, muscle and adipose tissues are the primary producers of heat in cold environments (Virtanen, 2014). Genes such as CKMT2, MYO1C, LCN9 and LCN15, are all related to energy metabolism. CKMT2 is responsible for the transfer of high energy phosphate from mitochondria to the cytosolic carrier (Qin et al., 1999). MYO1C is a widely expressed motor protein that links the actin cytoskeleton to cell membranes, acts as a slow transporter, and is associated with numerous cellular processes (Greenberg et al., 2012). The functions of LCN9 and LCN15 remain unknown, but another gene in the same Lipocalin superfamily (LCN2) is known for regulating thermogenic activation in adipose tissue (Flower, 2000; Guo et al., 2016). APOA5 is related to lipid transfer (Lim et al., 2014). CRHR2 plays an important role in white fat tissue loss and lipid metabolism under hypoxia (Xiong et al., 2014).

Another new discovery in this study was that many zinc finger protein genes were selected, such as ZNF259, ZNF365, ZNF384, ZNF410, ZNF646, ZNF668, and ZNF713. ZNF proteins play important roles in many biological processes (Cassandri et al., 2017).

In pathway analysis, only one SNARE interaction pathway in the vesicular transport pathway was identified; and this pathway included the VAMP1, STX4, STX6, and STX1B genes. VAMP1, a synaptobrevin family protein, is one of the main components of the SNARE complex. In mammalian cells, the SNARE complex consists of more than 60 member proteins (Salpietro et al., 2017). The SNARE complex drives membrane fusion (Zhang, 2017). However, it is unclear why this pathway was identified as under selection in the Min pig. This section pressure may be related to the high adaptability of this breed.

CONCLUSION

In this study, a total of 8,988,338 non-redundant SNPs plus 1,231,680 InDels were identified in Min pig. Both the PCA and phylogenetic tree results supported the notion that the genetic relationship of Min pig is between Asian pigs and European pigs. A total of 181 genes were subjected to selection pressure, especially some genes related to energy metabolism, such as CKMT2, MYO1C, LCN9, LCN15, APOA5 and CRHR2. In addition, many ZNF protein genes were also under selection pressure.

ACKNOWELDGEMENTS

This work was supported by grants China Agricultural Research System (CARS-36), Heilongjiang Outstanding Youth Fund (JQ2020C005), National Natural Science Foundation of Heilongjiang (LH2019C064) and Innovation Project of Heilongjiang Academy of Agricultural Sciences (2020FJZX007). We thank LetPub (www. letpub. com) for its linguistic assistance during the preparation of this manuscript.

Statement of conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Ai, H., Fang, X., Yang, B., Huang, Z., Chen, H., Mao, L., Zhang, F., Zhang, L., Cui, L., He, W., Yang, J., Yao, X., Zhou, L., Han, L., Li, J., Sun, S., Xie, X., Lai, B., Su, Y., Lu, Y., Yang, H., Huang, T., Deng, W., Nielsen, R., Ren, J. and Huang, L., 2015. Adaptation and possible ancient interspecies introgression in pigs identified by whole-genome sequencing. Nat. Genet., 47: 217-225. https://doi.org/10.1038/ng.3199

Barrow, A.D., Palarasah, Y., Bugatti, M., Holehouse, A.S., Byers, D.E., Holtzman, M.J., Vermi, W., Skjødt, K., Crouch, E. and Colonna, M., 2015. OSCAR is a receptor for surfactant protein D that activates TNF-α release from human CCR2+ inflammatory monocytes. J. Immunol., 194: 3317-3326. https://doi.org/10.4049/jimmunol.1402289

Bosse, M., Megens, H.J., Frantz, L.A., Madsen, O., Larson, G., Paudel, Y., Duijvesteijn, N., Harlizius, B., Hagemeijer, Y., Crooijmans, R.P. and Groenen, M.A., 2014. Genomic analysis reveals selection for Asian genes in European pigs following human-mediated introgression. Nat. Commun.5: 4392. https://doi.org/10.1038/ncomms5392

Cassandri, M., Smirnov, A., Novelli, F., Pitolli, C., Agostini, M., Malewicz, M., Melino, G. and Raschellà, G., 2017. Zinc-finger proteins in health and disease. Cell Death Discov., 3: 17071. https://doi.org/10.1038/cddiscovery.2017.71

China National Commission of Animal Genetics Resources. 2011. Animal genetics resources in China pigs. China Agriculture Press, Beijing.

Choi, J.W., Chung, W.H., Lee, K.T., Cho, E.S., Lee, S.W., Choi, B.H., Lee, S.H., Lim, W., Lim, D., Lee, Y.G., Hong, J.K., Kim, D.W., Jeon, H.J., Kim, J., Kim, N. and Kim, T.H., 2015. Whole-genome resequencing analyses of five pig breeds, including Korean wild and native, and three European origin breeds. DNA Res., 22: 259-267. https://doi.org/10.1093/dnares/dsv011

Choi, S.K., Lee, J.E., Kim, Y.J., Min, M.S., Voloshina, I., Myslenkov, A., Oh, J.G., Kim, T.H., Markov, N., Seryodkin, I., Ishiguro, N., Yu, L., Zhang, Y.P., Lee, H. and Kim, K.S., 2014. Genetic structure of wild boar (Sus scrofa) populations from East Asia based on microsatellite loci analyses. BMC Genet., 15: 85. https://doi.org/10.1186/1471-2156-15-85

D’Amours, O., Frenette, G., Bordeleau, L.J., Allard, N., Leclerc, P., Blondin, P. and Sullivan, R., 2012. Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine. Biol. Reprod., 87: 94. https://doi.org/10.1095/biolreprod.112.100990

Dogan, S., Vargovic, P., Oliveira, R., Belser, L.E., Kaya, A., Moura, A., Sutovsky, P., Parrish, J., Topper, E. and Memili, E., 2015. Sperm protamine-status correlates to the fertility of breeding bulls. Biol. Reprod., 92: 92. https://doi.org/10.1095/biolreprod.114.124255

Eibl, C., Grigoriu, S., Hessenberger, M., Wenger, J., Puehringer, S., Pinheiro, A.S., Wagner, R.N., Proell, M., Reed, J.C., Page, R., Diederichs, K. and Peti, W., 2012. Structural and functional analysis of the NLRP4 pyrin domain. Biochemistry, 51: 7330-7341. https://doi.org/10.1021/bi3007059

Flower, D.R., 2000. Beyond the superfamily: The lipocalin receptors. Biochim. biophys. Acta, 1482: 327-336. https://doi.org/10.1016/S0167-4838(00)00169-2

Giuffra, E., Kijas, J.M., Amarger, V., Carlborg, O., Jeon, J.T. and Andersson, L., 2000. The origin of the domestic pig: Independent domestication and subsequent introgression. Genetics, 154: 1785-1791.

Greenberg, M.J., Lin, T., Goldman, Y.E., Shuman, H. and Ostap, E.M., 2012. Myosin IC generates power over a range of loads via a new tension-sensing mechanism. Proc. natl. Acad. Sci. U. S. A., 109: 2433-2440. https://doi.org/10.1073/pnas.1207811109

Groenen, M.A., Archibald, A.L., Uenishi, H., Tuggle, C.K., Takeuchi, Y., Rothschild, M.F., Rogel-Gaillard, C., Park, C., Milan, D., Megens, H.J., Li, S., Larkin, D.M., Kim, H., Frantz, L.A., Caccamo, M., Ahn, H., Aken, B.L., Anselmo, A., Anthon, C., Auvil, L., Badaoui, B., Beattie, C.W., Bendixen, C., Berman, D., Blecha, F., Blomberg, J., Bolund, L., Bosse, M., Botti, S., Bujie, Z., Bystrom, M., Capitanu, B., Carvalho-Silva, D., Chardon, P., Chen, C., Cheng, R., Choi, S.H., Chow, W., Clark, R.C., Clee, C., Crooijmans, R.P., Dawson, H.D., Dehais, P., De Sapio, F., Dibbits, B., Drou, N., Du, Z.Q., Eversole, K., Fadista, J., Fairley, S., Faraut, T., Faulkner, G.J., Fowler, K.E., Fredholm, M., Fritz, E., Gilbert, J.G., Giuffra, E., Gorodkin, J., Griffin, D.K., Harrow, J.L., Hayward, A., Howe, K., Hu, Z.L., Humphray, S.J., Hunt, T., Hornshøj, H., Jeon, J.T., Jern, P., Jones, M., Jurka, J., Kanamori, H., Kapetanovic, R., Kim, J., Kim, J.H., Kim, K.W., Kim, T.H., Larson, G., Lee, K., Lee, K.T., Leggett, R., Lewin, H.A., Li, Y., Liu, W., Loveland, J.E., Lu, Y., Lunney, J.K., Ma, J., Madsen, O., Mann, K., Matthews, L., McLaren, S., Morozumi, T., Murtaugh, M.P., Narayan, J., Nguyen, D.T., Ni, P., Oh, S.J., Onteru, S., Panitz, F., Park, E.W., Park, H.S., Pascal, G., Paudel, Y., Perez-Enciso, M., Ramirez-Gonzalez, R., Reecy, J.M., Rodriguez-Zas, S., Rohrer, G.A., Rund, L., Sang, Y., Schachtschneider, K., Schraiber, J.G., Schwartz, J., Scobie, L., Scott, C., Searle, S., Servin, B., Southey, B.R., Sperber, G., Stadler, P., Sweedler, J.V., Tafer, H., Thomsen, B., Wali, R., Wang, J., Wang, J., White, S., Xu, X., Yerle, M., Zhang, G., Zhang, J., Zhang, J., Zhao, S., Rogers, J., Churcher, C. and Schook, L.B., 2012. Analyses of pig genomes provide insight into porcine demography and evolution. Nature, 491, 393-398. https://doi.org/10.1038/nature11622

Guo, H., Foncea, R., O’Byrne, S.M., Jiang, H., Zhang, Y., Deis, J.A., Blaner, W.S., Bernlohr, D.A. and Chen, X., 2016. Lipocalin 2: Aa regulator of retinoid homeostasis and retinoid-mediated thermogenic activation in adipose tissue. J. biol. Chem., 291: 11216-11229. https://doi.org/10.1074/jbc.M115.711556

Guo, Y., Hou, L., Zhang, X., Huang, M., Mao, H., Chen, H., Ma, J., Chen, C., Ai, H., Ren, J. and Huang, L., 2015. A meta analysis of genome-wide association studies for limb bone lengths in four pig populations. BMC Genet., 19: 65. https://doi.org/10.1186/s12863-015-0257-1

Ishizaki, F., Nishiyama, T., Kawasaki, T., Miyashiro, Y., Hara, N., Takizawa, I., Naito, M. and Takahashi, K., 2013. Androgen deprivation promotes intratumoral synthesis of dihydrotestosterone from androgen metabolites in prostate cancer. Sci. Rep., 3: 1528. https://doi.org/10.1038/srep01528

Jin, X., Chen, Y.H., Liu, Z., Deng, Y., Li, N.N., Huang, H., Qi, M., Yi, X. and Zhu, J., 2015. Identification of copy number variation in the gene for autosomal dominant optic atrophy, OPA1, in a Chinese pedigree. Genet. Mol. Res., 14: 10961-10972. https://doi.org/10.4238/2015.September.21.8

Kirthika, P., Ali, M.A., Behera, P., Subudhi, P.K., Tolenkhomba, T.C, and Gali, J.M., 2017. Dynamics of cytokine gene expression in peripheral blood mononuclear cells of indigenous and exotic breeds of pigs in India. Anim. Sci. J., 88: 1794-1800. https://doi.org/10.1111/asj.12827

Li, M., Tian, S., Jin, L., Zhou, G., Li, Y., Zhang, Y., Wang, T., Yeung, C.K., Chen, L., Ma, J., Zhang, J., Jiang, A., Li, J., Zhou, C., Zhang, J., Liu, Y., Sun, X., Zhao, H., Niu, Z., Lou, P., Xian, L., Shen, X., Liu, S., Zhang, S., Zhang, M., Zhu, L., Shuai, S., Bai, L., Tang, G., Liu, H., Jiang, Y., Mai, M., Xiao, J., Wang, X., Zhou, Q., Wang, Z., Stothard, P., Xue, M., Gao, X., Luo, Z., Gu, Y., Zhu, H., Hu, X., Zhao, Y., Plastow, G.S., Wang, J., Jiang, Z., Li, K., Li, N., Li, X. and Li, R., 2013. Genomic analyses identify distinct patterns of selection in domesticated pigs and Tibetan wild boars. Nat. Genet., 45: 1431-1438. https://doi.org/10.1038/ng.2811

Lim, H.H., Choi, M., Kim, J.Y., Lee, J.H. and Kim, O.Y., 2014. Increased risk of obesity related to total energy intake with the APOA5-1131T > C polymorphism in Korean premenopausal women. Nutr. Res., 34: 827-836. https://doi.org/10.1016/j.nutres.2014.08.018

Liu, G., Wang, Y., Jiang, S., Sui, M., Wang, C., Kang, L., Sun, Y. and Jiang, Y., 2019. Suppression of lymphocyte apoptosis in spleen by CXCL13 after porcine circovirus type 2 infection and regulatory mechanism of CXCL13 expression in pigs. Vet. Res.50: 17. https://doi.org/10.1186/s13567-019-0634-2

Liu, Y., Yang, X., Jing, X., He, X., Wang, L., Liu, Y. and Liu, D., 2017. Transcriptomics analysis on excellent meat quality traits of skeletal muscles of the Chinese indigenous Min Pig compared with the large white breed. Int. J. mol. Sci., 19: pii:E21. https://doi.org/10.3390/ijms19010021

Ma, Y., Wei, J., Zhang, Q., Chen, L., Wang, J., Liu, J. and Ding, X.A., 2015. Genome scan for selection signatures in pigs. PLoS One, 10: e0116850. https://doi.org/10.1371/journal.pone.0116850

Monahan, K. and Lomvardas, S., 2015. Monoallelic expression of olfactory receptors. Ann. Rev. Cell Dev. Biol., 31: 721-740. https://doi.org/10.1146/annurev-cellbio-100814-125308

Qin, W., Khuchua, Z., Boero, J., Payne, R.M. and Strauss, A.W., 1999. Oxidative myocytes of heart and skeletal muscle express abundant sarcomeric mitochondrial creatine kinase. Histochem. J., 31: 357-365. https://doi.org/10.1023/A:1003748108062

Ren, D.R., Ren, J., Ruan, G.F., Guo, Y.M., Wu, L.H., Yang, G.C., Zhou, L.H., Li, L., Zhang, Z.Y. and Huang, L.S., 2012. Mapping and fine mapping of quantitative trait loci for the number of vertebrae in a white Duroc×Chinese Erhualian intercross resource population. Anim. Genet., 43: 545-551. https://doi.org/10.1111/j.1365-2052.2011.02313.x

Ruan, L., Zhang, Z., Mu, L., Burnley, P., Wang, L., Coder, B., Zhuge, Q. and Su, D.M., 2014. Biological significance of FoxN1 gain-of-function mutations during T and B lymphopoiesis in juvenile mice. Cell Death Dis., 5: e1457. https://doi.org/10.1038/cddis.2014.432

Salpietro, V., Lin, W., Delle-Vedove, A., Storbeck, M., Liu, Y., Efthymiou, S., Manole, A., Wiethoff, S., Ye, Q., Saggar, A., McElreavey, K., Krishnakumar, S.S., Synaps Study Group, Pitt, M., Bello, O.D., Rothman, J.E., Basel-Vanagaite, L., Hubshman, M.W., Aharoni, S., Manzur, A.Y., Wirth, B. and Houlden, H., 2017. Homozygous mutations in VAMP 1 cause a presynaptic congenital myasthenic syndrome. Annls. Neurol., 81:597-603. https://doi.org/10.1002/ana.24905

Shankar, S., Chua, J.Y., Tan, K.J., Calvert, M.E., Weng, R., Ng, W.C., Mori, K. and Yew, J.Y., 2015. The neuropeptide tachykinin is essential for pheromone detection in a gustatory neural circuit. Elife, 4: e06914. https://doi.org/10.7554/eLife.06914

Srinivasan, S., Chitalia, V., Meyer, R.D., Hartsough, E., Mehta, M., Harrold, I., Anderson, N., Feng, H., Smith, L.E., Jiang, Y., Costello, C.E. and Rahimi, N., 2015. Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis. Angiogenesis, 18: 449-462. https://doi.org/10.1007/s10456-015-9468-3

Tadaki, H., Saitsu, H., Nishimura-Tadaki, A., Imagawa, T., Kikuchi, M., Hara, R., Kaneko, U., Kishi, T., Miyamae, T., Miyake, N., Doi, H., Tsurusaki, Y., Sakai, H., Yokota, S. and Matsumoto, N., 2011. De novo 19q1342 duplications involving NLRP gene cluster in a patient with systemic-onset juvenile idiopathic arthritis. J. Hum. Genet., 56: 343-347. https://doi.org/10.1038/jhg.2011.16

Traspov, A., Deng, W., Kostyunina, O., Ji, J., Shatokhin, K., Lugovoy, S., Zinovieva, N., Yang, B. and Huang, L., 2016. Erratum to: Population structure and genome characterization of local pig breeds in Russia, Belorussia, Kazakhstan and Ukraine. Genet. Select. Evol., 48: 57. https://doi.org/10.1186/s12711-016-0235-8

Vilagran, I., Castillo, J., Bonet, S., Sancho, S., Yeste, M., Estanyol, J.M. and Oliva, R., 2013. Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity. Theriogenology, 80: 443-450. https://doi.org/10.1016/j.theriogenology.2013.05.006

Virtanen, K.A., 2014. BAT thermogenesis: Linking shivering to exercise. Cell Metab., 19: 352-354. https://doi.org/10.1016/j.cmet.2014.02.013

Wang, K., Wu, P., Yang, Q., Chen, D., Zhou, J., Jiang, A., Ma, J., Tang, Q., Xiao, W., Jiang, Y., Zhu, L., Li, X. and Tang, G., 2018. Detection of selection signatures in Chinese Landrace and Yorkshire pigs based on genotyping-by-sequencing data. Front. Genet., 9: 119. https://doi.org/10.3389/fgene.2018.00119

Wang, Z., Sun, H., Chen, Q., Zhang, X., Wang, Q. and Pan, Y., 2019. A genome scan for selection signatures in Taihu pig breeds using next-generation sequencing. Animal13: 683-693. https://doi.org/10.1017/S1751731118001714

Xiang, H., Gao, J., Cai, D., Luo, Y., Yu, B., Liu, L., Liu, R., Zhou, H., Chen, X., Dun, W., Wang, X., Hofreiter, M. and Zhao, X., 2017. Origin and dispersal of early domestic pigs in northern China. Sci. Rep., 7: 5602. https://doi.org/10.1038/s41598-017-06056-8

Xiong, Y., Qu, Z., Chen, N., Gong, H., Song, M., Chen, X., Du, J. and Xu, C., 2014. The local corticotropin- releasing hormone receptor 2 signalling pathway partly mediates hypoxia-induced increases in lipolysis via the cAMP-protein kinase Asignalling pathway in white adipose tissue. Mol. Cell. Endocrinol., 392: 106-114. https://doi.org/10.1016/j.mce.2014.05.012

Xu, Q., Liu, X., Chao, Z., Wang, K., Wang, J., Tang, Q., Luo, Y., Zheng, J., Tan, S. and Fang, M., 2019. Transcriptomic analysis of coding genes and non-coding RNAs reveals complex regulatory networks underlying the black back and white belly coat phenotype in Chinese Wuzhishan pigs. Genes (Basel), 10: pii: E201. https://doi.org/10.3390/genes10030201

Yang, L., Güell, M., Niu, D., George, H., Lesha, E., Grishin, D., Aach, J., Shrock, E., Xu, W., Poci, J., Cortazio, R., Wilkinson, R.A., Fishman, J.A. and Church, G., 2015. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science, 350: 1101-1104. https://doi.org/10.1126/science.aad1191

Young, L.D., 1992. Effects of Duroc, Meishan, Fengjing, and Minzhu boars on carcass traits of first-cross barrows. J. Anim. Sci., 70: 2030-2037. https://doi.org/10.2527/1992.7072030x

Zhang, B., Ban, D., Gou, X., Zhang, Y., Yang, L., Chamba, Y. and Zhang, H., 2019. Genome-wide DNA methylation profiles in Tibetan and Yorkshire pigs under high-altitude hypoxia. J. Anim. Sci. Biotechnol., 10: 25. https://doi.org/10.1186/s40104-019-0316-y

Zhang, Y., 2017. Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers. Protein Sci., 26: 1252-1265. https://doi.org/10.1002/pro.3116

Zhao, X., Cheng, G., Yan, W., Liu, M., He, Y., Zheng, Z., 2009. Characterization and virus-induced expression profiles of the porcine interferon-omega multigene family. J. Interf. Cytok. Res., 29: 687-693. https://doi.org/10.1089/jir.2008.0060

To share on other social networks, click on any share button. What are these?

Pakistan Journal of Zoology

August

Vol. 54, Iss. 4, Pages 1501-2001

Featuring

Click here for more

Subscribe Today

Receive free updates on new articles, opportunities and benefits


Subscribe Unsubscribe