| Shandong Province has rich and precious indigenous pig breeds,and its germplasm characteristics are high-stress resistance,strong fecundity,delicious meat quality,rough feeding resistance,and cold tolerance.To clarify the molecular genetic mechanism of these excellent germplasm characteristics is the focus of current pig genetic breeding work.In addition,a large number of Western pig breeds have been introduced and hybridized,which seriously affected the purity of Shandong indigenous pig breeds and the preservation of germplasm characteristics.Therefore,a comprehensive understanding of the genetic diversity of Shandong indigenous pig breeds and revealing the genetic relationship with other pig breeds is an important prerequisite for the conservation and rational utilization of biodiversity of germplasm resources.With the application of high-throughput and low-cost DNA sequencing technology,it is possible to study the genetic variation of indigenous pig breeds from the whole genome.In this study,SLAF-seq was used to analyze the genetic differences between Shandong indigenous pig breeds and Western pig breeds,and to study the function of key candidate genes.The specific research content and results were as follows:(1)Genome-wide genetic variation detection.A total of 80 samples from seven Shandong indigenous pig breeds(Zaozhuang Heigai,Dapulian,Wulian Black,Laiwu,Yantai Laizhou Black,Yantai Wendeng Black,Licha Black)and three introduced pig breeds(Duroc,Large White,Landrace)were sequenced by SLAF-seq to construct genomic libraries and develop genome-wide molecular markers.After quality control,a total of 1010652 SLAF tags were obtained,with an average sequencing depth of 16.67×.The sequencing data and the northern wild boar data in the database were combined for SNP marker development,and a total of314243 representative SNPs were identified.(2)Genetic diversity and population structure analysis.The genetic diversity and population structure of Shandong indigenous pig breeds were analyzed by using the identified genetic variation.The analysis of genetic diversity mainly includes six parameters.The results showed that the genetic diversity of Shandong indigenous pig breeds was higher than that of introduced pig breeds.Zaozhuang Heigai pigs had the highest genetic diversity(Ho = 0.2926,He = 0.3752,Nei = 0.3814,PIC = 0.2988,and MAF = 0.2852),whereas Landrace had the lowest genetic diversity(Ho = 0.2283,He = 0.3259,Nei = 0.3511,PIC = 0.2639,and MAF =0.2378).The nucleotide polymorphism(?)value was ranged from 0.00002820 to 0.00003722 as compared to 0.00002067 to 0.00002589 of Western pig breeds.Secondly,the evaluation results of the degree of differentiation among populations showed that the index of population differentiation between Wulian black pig and Yantai Wendeng black pig(Fst=0.0831)was the lowest and that between Laiwu pig and Duroc pig was the highest(Fst=0.5134).The results of the phylogenetic tree,principal component analysis,and population structure analysis showed that the Shandong indigenous pig breeds can be clearly and independently clustered,and the genetic relationship among populations is closer than that of the introduced pig breeds.(3)Selective sweep analysis.After a long period of natural or artificial selection,Shandong indigenous pig breeds have developed characteristics such as good meat quality and strong disease resistance,so there may be imprints of these selection effects in their genomes.We detected and analyzed the whole genome selection signals of indigenous pig breeds.The results showed that 162 and 157 differentially selected regions were found in Dapulian and Laiwu pig populations respectively.A total of 841 positive selection genes were screened from Dapulian pigs.The results of the functional analysis showed that these genes were related to immunity(TNFRST8,RAB5 B,ADAM17,and GNLY)and diseases(SBNO2,IFNLR1,and URI1).For example,GNLY was related to antifungal and innate immunity and IFNLR1 was related to cytokine activity and antiviral.These genes may have a potential relationship with the traits of wide adaptability and strong disease resistance of Dapulian pigs.A total of707 selected candidate genes were distributed in the differentially selected regions from Laiwu pigs which were related to muscle growth(DMD,SKIL,EYA1)and fat deposition(FITM1,ADRA2 A,NR4A1).Among them,DMD was associated with the development of muscle fiber and cytoskeleton and FITM1 was lipid droplet deposition related.(4)Function of IFNLR1 in PRRSV infection of PAM cells.In this study,the complete coding sequence(CDS)region of porcine IFNLR1 gene was cloned for the first time and analyzed by bioinformatics.The complete CDS region of porcine IFNLR1 gene was 1578 bp in length(MT741991),which encoded a protein containing 525 amino acids.The phylogenetic tree analysis showed that it was closely related to Camels ferus and Vicugna pacos.The tissue distribution of IFNLR1 gene in Dapulian and Landrace pigs was analyzed by q RT-PCR,and the results showed that there were differences in IFNLR1 gene expression differential expression between species and tissues.To understand the immunological characteristics of porcine IFNLR1,a highly pathogenic porcine reproductive and respiratory syndrome virus strain was used to infect porcine alveolar macrophages.The results showed that the transcription level of IFNLR1 m RNA was significantly down-regulated,while the m RNA expression level of IFN-?1 and IFN-?3 was significantly up-regulated after PRRSV infection.The above results indicate that type III interferon pathway plays an important role in the process of PRRSV infection of PAM cells.To further clarify its biological function,we constructed pc DNA3.1(+)-IFNLR1 overexpression plasmid and synthesized si-IFNLR1 interference fragment respectively.The results showed that overexpression of porcine IFNLR1 can significantly inhibit the replication of PRRSV,up-regulate the m RNA levels of interferon-stimulated genes and antiviral genes,and down-regulate the expression of some inflammatory cytokines.Overexpression of IFNLR1 significantly inhibited the proliferation of PAM cells infected with PRRSV and induced the expression of p-STAT1.The opposite result was observed after IFNLR1 gene was depressed.When JAK/STAT pathway was inhibited,its ability to inhibit cell proliferation disappeared.Flow cytometry test results indicated that the proportion of G2/M phase of PAM cells infected with PRRSV increased significantly.The proportion of G0/G1 and S phase of IFNLR1 overexpression cells increased significantly,and the proportion of G2/M phase decreased.When JAK inhibitor was added,the proportion of PAM cells overexpressing IFNLR1 in G2/M phase increased to a certain extent.In conclusion,this study explored the genetic diversity and genetic relationship between Shandong indigenous pig breeds and Western pig breeds,and found that Shandong indigenous breeds exhibit high genetic diversity,which provided valuable theoretical basis for the development and utilization of Shandong indigenous pigs.Porcine IFNLR1 gene participated in immunoregulation in the process of PAM cells resisting PRRSV infection,and it was differentially expressed in Dapulian and Landrace pigs,which can be used as a candidate gene for high disease resistance of indigenous pig breeds. |
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