Scan of the endogenous retrovirus sequences across the swine genome and survey of their copy number variation and sequence diversity among various Chinese and Western pig breeds.

In pig-to-human xenotransplantation, the transmission risk of porcine endogenous retroviruses (PERVs) is of great concern. However, the distribution of PERVs in pig genomes, their genetic variation among Eurasian pigs, and their evolutionary history remain unclear. We scanned PERVs in the current pig reference genome (assembly Build 11.1), and identified 36 long complete or near-complete PERVs (lcPERVs) and 23 short incomplete PERVs (siPERVs). Besides three known PERVs (PERV-A, -B, and -C), four novel types (PERV-JX1, -JX2, -JX3, and -JX4) were detected in this study. According to evolutionary analyses, the newly discovered PERVs were more ancient, and PERV-Bs probably experienced a bottleneck ~0.5 million years ago (Ma). By analyzing 63 high-quality porcine whole-genome resequencing data, we found that the PERV copy numbers in Chinese pigs were lower (32.0±4.0) than in Western pigs (49.1±6.5). Additionally, the PERV sequence diversity was lower in Chinese pigs than in Western pigs. Regarding the lcPERV copy numbers, PERV-A and -JX2 in Western pigs were higher than in Chinese pigs. Notably, Bama Xiang (BMX) pigs had the lowest PERV copy number (27.8±5.1), and a BMX individual had no PERV-C and the lowest PERV copy number (23), suggesting that BMX pigs were more suitable for screening and/or modification as xenograft donors. Furthermore, we identified 451 PERV transposon insertion polymorphisms (TIPs), of which 86 were shared by all 10 Chinese and Western pig breeds. Our findings provide systematic insights into the genomic distribution, variation, evolution, and possible biological function of PERVs.

Identification and functional study of a shrimp Dorsal homologue.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here, identification and functional analysis of LvDorsal, a Dorsal homologue from the Pacific white shrimp Litopenaeus vannamei, were described. The full-length cDNA of LvDorsal is 2204bp with an open reading frame that encodes 400 amino acids. The deduced LvDorsal contains a conserved Rel homology domain (RHD), an IPT (Ig-like, plexins and transcription factors) domain and a nucleus localization signal, suggesting that it belongs to the class II NF-kappaB. RT-PCR analysis showed that LvDorsal mRNAs were expressed in all the tissues tested, including gill, epidermis, hemocytes, intestine, stomach, eyestalk, brain, hepatopancreas, muscle, heart and pyloric caecum. Immunofluorescence assay showed that recombinant LvDorsal was translocated into the nucleus of Drosophila S2 cells. Electrophoretic mobility shift assay illustrated that recombinant LvDorsal RHD from S2 cells bound specifically with D. melanogaster kappaB motifs. Additionally, the dual-luciferase reporter assays indicated that LvDorsal could transactivate the reporter gene controlled by the 5' flanking region of shrimp penaeidin-4 and Drosophila attacin genes, suggesting that LvDorsal can regulate the transcription of shrimp penaeidin-4 gene. Study of LvDorsal will help us to better understand shrimp immunity and may help to obtain more effective methods to prevent shrimp diseases.

Identification and functional study of a shrimp Relish homologue.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here we describe the identification and functional analysis of a Relish homologue, LvRelish and its shorter isoform sLvRelish, from the Pacific white shrimp, Litopenaeus vannamei. The LvRelish gene has 22 exons in approximately 15 kb genomic sequence. The full-length cDNA of LvRelish is 4071 bp with an open reading frame that encodes 1207 amino acids. LvRelish contains a conserved Rel homology domain (RHD), a nucleus localization signal, an IkappaB-like domain (six ankyrin repeats), and a death domain, suggesting that it belongs to the class I NF-kappaB. sLvRelish cDNA is 1051 bp encoding 317 amino acids. It shares the RHD region with LvRelish. RT-PCR analysis showed that LvRelish and sLvRelish mRNAs were expressed at different levels in tissues. Western blot analysis showed that recombinant intact LvRelish could be cleaved into two fragments in S2 cells, and immunofluorescence assay showed that the plasmid-expressed LvRelish protein was seen both in the cytoplasm and the nucleus. Electrophoretic mobility shift assay showed that recombinant RHD of LvRelish in S2 cells bound specifically with Drosophila melanogaster kappaB motifs in vitro. Both the LvRelish and its RHD domain transactivated the reporter gene controlled by the 5' flanking region of penaeidin 4, an antibacterial peptide of shrimp, suggesting that LvRelish can regulate the transcription of penaeidin 4 gene. Identification of LvRelish will help us better understand shrimp immunity and may help obtain more effective methods to prevent shrimp diseases.

An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes.

Invertebrates rely on innate immunity as the first line defense against microbes. In Drosophila, the inducible antimicrobial peptides (AMPs) regulated by the Toll and immune deficiency (Imd) pathways are important effectors in innate immunity. Here we report an immune deficiency homolog (LvIMD) from the white shrimp, Litopenaeus vannamei. The full-length cDNA of LvIMD is 758 bp with an open reading frame of 483 bp that encodes a putative protein of 160 amino acids including a death domain at the C-terminus. LvIMD death domain shows similarity to that of Drosophila IMD and human receptor interacting protein 1 (RIP1) of the tumor necrosis factor receptor (TNFR) pathway, with 27.9% and 26.4% identity, respectively. Phylogenetic analysis shows that LvIMD clusters with a predicted protein from the starlet sea anemone (Nematostella vectensis) independent to insect IMDs and vertebrates RIP1s. LvIMD mRNA is expressed in most tissues and is induced in hepatopancreas and hemocytes after immune challenge. Luciferase reporter assays confirm that LvIMD is able to induce the expression of AMP genes, including Drosophila Attacin A and shrimp Penaeidin 4 in S2 cells. To our knowledge, this is the first report that LvIMD participates in innate signaling to activate the expression of AMP genes in shrimp.

A novel prophenoloxidase 2 exists in shrimp hemocytes.

The prophenoloxidase (proPO)-activating system in crustaceans and other arthropods is regarded as a constituent of the immune system and plays an important role in defense against pathogens. Hitherto in crustaceans, only one proPO gene per species has been identified. Here we report the identification of a novel proPO-2 (LvproPO-2) from the hemocytes of Litopenaeus vannamei, which shows 72% identity to proPO-1 (LvproPO-1) cloned previously. Northern blotting analysis and quantitative real-time PCR reveal that LvproPO-2 is mainly expressed in the hemocytes, and its expression is down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Western blotting analysis shows that most LvproPO-2/LvPO-2 (L. vannamei phenoloxidase-2) exists in the hemocytes, but not in plasma of L. vannamei. LvproPO-2/LvPO-2 could be detected on the hemocyte surface and the nucleus of hemocytes by indirect immunofluorescence assay (IFA). These findings provide insight into the molecular biological basis for further studying on the defense mechanism of shrimp innate immunity, especially on the proPO-activating system and melanization cascade of shrimp.

Characterization of a prophenoloxidase from hemocytes of the shrimp Litopenaeus vannamei that is down-regulated by white spot syndrome virus.

Previously, a prophenoloxidase (proPO) gene (named proPO-a here) from hemocytes of Litopenaeus vannamei was isolated. Here, a proPO-b gene was also identified and characterized from hemocytes of L. vannamei. The cDNA sequences of proPO-a and proPO-b were compared, and it was found that both proPOs had a microsatellite DNA site near the 3' end of the open reading frame (ORF). However, the microsatellite DNA of proPO-b contained a compound imperfect simple sequence repeats (SSR) ((CT)(38)(CA)(8)(AA)(CA)(3)(TA)(CA)(14)), which was different from the perfect one ((CT)(20)) of proPO-a, and the cDNA sequences of proPO-a and proPO-b prior to the microsatellite DNA were almost identical, but differed after the microsatellite DNA. ProPO-b (3232 bp) was longer than proPO-a (2471 bp). The 3' UTR sequence after SSR of proPO-a was not detected in shrimp randomly collected from five different geographically separate populations by reverse-transcription polymerase chain reaction (RT-PCR). On the contrary, the 3' UTR sequence of proPO-b was detected in all five groups of shrimps. Northern blot analysis showed that a transcript at approximately 3.2kb, but not 2.5kb, was detected mainly in hemocytes, and also present in midgut, gill, heart, stomach, posterior midgut cecum, and cuticular epidermis, but no signal was detected in hepatopancreas and musculature. RT-PCR and quantitative real-time RT-PCR analysis showed similar results of the proPO-b expression profile in these shrimp tissues. We also observed that proPO-b expression was down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Our results suggest that proPO-b is a main transcript form of proPO gene in L. vannamei, and it may play a role in defence against WSSV virus.

Genetic variations of the porcine PRKAG3 gene in Chinese indigenous pig breeds.

Four missense substitutions (T30N, G52S, V199I and R200Q) in the porcine PRKAG3 gene were considered as the likely candidate loci affecting meat quality. In this study, the R200Q substitution was investigated in a sample of 62 individuals from Hampshire, Chinese Min and Erhualian pigs, and the genetic variations of T30N, G52S and V199I substitutions were detected in 1505 individuals from 21 Chinese indigenous breeds, 5 Western commercial pig breeds, and the wild pig. Allele 200R was fixed in Chinese Min and Erhualian pigs. Haplotypes II-QQ and IV-QQ were not observed in the Hampshire population, supporting the hypothesis that allele 200Q is tightly linked with allele 199V. Significant differences in allele frequencies of the three substitutions (T30N, G52S and V199I) between Chinese indigenous pigs and Western commercial pigs were observed. Obvious high frequencies of the "favorable" alleles 30T and 52G in terms of meat quality were detected in Chinese indigenous pigs, which are well known for high meat quality. However, the frequency of the "favorable" allele 199I, which was reported to have a greater effect on meat quality in comparison with 30T and 52G, was very low in all of the Chinese indigenous pigs except for the Min pig. The reasons accounting for this discrepancy remain to be addressed. The presence of the three substitutions in purebred Chinese Tibetan pigs indicates that the three substitutions were ancestral mutations. A novel A/G substitution at position 51 in exon 1 was identified. The results suggest that further studies are required to investigate the associations of these substitutions in the PRKAG3 gene with meat quality of Chinese indigenous pigs, and to uncover other polymorphisms in the PRKAG3 gene with potential effects on meat quality in Chinese indigenous pigs.

[Studies of the relationship of melanocortin receptor 1(MC1R) gene with coat color phenotype in pigs].

Although coat color in pigs has no direct relation with economic traits, it affects economic benefit significantly, coat color selection are widely used in pig breeding and production. PCR-Acc II-RFLP, PCR-BspH I-RFLP and PCR-SSCP were used in combination to analyze genotype at MC1R locus among individuals from 16 full-sib pedigrees and 6 Chinese native breeds including Jinhua, Jiaxing Black, Yushan Black, Leping Spotted, Shanggao Spotted and Shengxian Spotted pig. It was found that the Chinese native pig breeds carry a dominant black allele at MC1R at high frequency, this ED1 allele was suggested to be the major allele controlling black coat color in Chinese native pig breed. In addition, the evidence for a new allele was obtained in Shengxian Spotted pigs by PCR-SSCP analysis. It was reconfirmed from the result of pedigree analysis that ED1 was dominant over EP and e, while EP was incompletely dominant over e.

Research on the genetic variations of a1-fucosytransferase (FUT1) gene in 26 pig breeds.

Enterotoxigenic Escherichia coli F18(ECF18) is a main pathogen that causes edema disease and post-weaning diarrhoea in piglets, and al-fucosytransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the receptor for ECF18 bacteria. The genetic variations at position 307 nucleotide in open reading frame of FUT1 gene in 26 pig breeds (total 1458 individuals) from 5 western commercial pig breeds and 21 Chinese native pig breeds were investigated by PCR-RFLP. The results showed that the genetic polymorphisms of the FUT1 locus were only detected in 5 western pig breeds and the Chinese Lingao pig breed, 5 western pig breeds possessed 3 different genotypes, and Lingao pig breed had two susceptible genotypes GG and AG, while all the other 20 Chinese native pig breeds only presented the susceptible genotype GG. The results indicated that if M307G-A point mutation in the coding region of FUT1 gene was the key factor determining the expression of the ECF18 receptor, most of Chinese native pig breeds were absent of the genetic background on the resistance to ECF18 bacteria. In this case, it was inferred that the resistance gene to ECF18 might be originated from western pig breeds. In addition, it is of great importance for the conservation of Lingao pig breed as it is the only found Chinese native pig breed possessing resistance M307A allele in FUT1 gene. Generally, compared with exotic pig breeds, Chinese native pig breeds have stronger resistance to edema disease and post-weaning diarrhoea in piglets. The results suggested that further study should be done to identify and characterize putative QTL (quantitative trait locus) or/and the functional gene responsible for the resistance to ECF18 in Chinese native pig breeds.

[Polymorphism analysis of porcine myogenin gene by PCR-RFLP].

The polymorphisms of porcine myogenin gene in 561 pigs including Duroc, Landrace, Large Yorkshire, Nanchang white pig, Erhualian, Meishan, Yushan black pig, Leping spotted pig, Jinhua black head-hind pig and Shanggao black head-hind pig were detected by PCR-RFLPs with three different pairs of primers,and the PCR products were digested by MspI. The results showed that most of the Duroc, Landrace, Large Yorkshire, Nanchang white pigs presented as AA genotype, while more animals of the six Chinese local pig breeds except for Leping spotted pig presented as BB genotype at PCR1 MspI-RFLP site. The six Chinese local breeds presented as MM genotype except that one Yushan black pig presented as MN genotype, while more swines of the exotic breeds including Duroc, Landrace, Large Yorkshire presented as NN genotype, and Nanchang white pig appeared to be closer to the exotic breeds at PCR2 MspI-RFLP site. PCR product was obtained in all the swine by PCR3, but the MspI restriction site was not found in the tested pig breeds including Meishan and blood closely related Erhualian pig.

[Relationship of growth hormone (GH 2) genotypes with some production performances in pig].

The genotypes of 117 Nanchang White pigs and 361 Large Yorkshire pigs at GH 2 locus were detected by PCR-RFLP. The PCR products were cut by Apa I, and produced two alleles: A(449 + 101 + 55 bp), and B(316 + 133 + 101 + 49 bp). Effects of different genotypes on some important production traits involving the birth weight, 2-month body weight, 6-month body weight, corrected back-fat thickness, average back-fat thickness, feed to gain ratio and lean percentage were analyzed. The results showed that in Nanchang White pigs, no significant differences were observed between different genotypes and different growth and carcass traits; while in Large Yorkshire, the pigs with BB genotype had more lean percentage than pigs with AA genotype (P < 0.05).

[Studies of population genetic relationships among 24 Chinese and exotic pig breeds using AFLP analysis].

A total of 12 AFLP primer combinations were used to detect genetic variation of pooled DNA in a sample of 19 Chinese native pig breeds, 1 cultivated pig breed and 4 European and American pig breeds. The genetic similarity coefficient of 24 pig beeds was calculated from AFLP data, UPGMA cluster analysis was also performed. The 12 primer combinations generated more than 1000 bands, of which 208 bands were polymorphic, 17.3 polymorphic markers were detected by one primer combination on the average. Thirteen putative breed specific bands were produced in the pooled DNA of 8 pig breeds. The cultivated pig breed and 4 exotic pig breeds were clustere into one group, while 19 Chinese native pig breeds were gathered into the other group in the UPGMA tree. The result indicated that AFLP analysis had high assay efficiency index (Ai) and provided a valuable tool for assaying genetic diversity and breed characterization in pigs. Chinese native pig breeds and exotic pig breeds show remarkable genetic differentiation, which had farther genetic relationships. Nanchang White pig and Large White pig, Yushan Black pig and Yanshan Black pig had intimate genetic relationships with each other respectively, which were consistent with its breeding history, geographical distribution and RAPD analysis results. In addition, the reasons for cluster results of some pig breeds from AFLP data were not consistent with morphology, geographical distribution and existing classification were discussed.