| Ningxiang pigs,a fat-type pig,are native to Ningxiang County in Hunan Province,with thousands of years of breeding history.In the long-term breeding process,Ningxiang pigs,one of the four famous pig species in China,are popular for their advantages including tender and succulent meat,unique flavor,and high-quality unsaturated fatty acids.A large number of studies have showed that the genetic diversity of local Chinese breeding pigs is higher than that of European pigs,and there are many differences in fat deposition,capacity of fat deposition and fatty acid content among different pig breeds.However,the genetic characteristics of Ningxiang pig have not been systematically studied.The development of long-read sequencing and genome assembly provide an opportunity to improve and update pig genomes and reveal the full range of structural variations between the local Chinese and European pig breeds.Blood samples were obtained from a purebred mature female of Ningxinag pig provided by the Ningxiang pig original breeding pig farm.Here we aimed to sequence and assemble a highly contiguous Ningxiang pig genome using single-molecule real-time sequencing,next-generation mapping,Bionano map,and Hi-C approaches.The assembly had a genome size of 2.438 Gb,which had 32.16 Mb and 357 in Contig N50 and contig number respectively,with 20,912 annotated protein-coding genes and 34.04% repetitive sequences.The optical map was constructed by BioNano and 19 chromosomes was built by Hi-C.The Ningxiang pig genome compared to Duroc pig with third-generation genome sequencing,the representative lean type breed,could provide more different information.Compared to the current Duroc pig genome,our assembly increased sequence contiguity.According to Duroc genome coordinates,in total,26673 SVs were annotated in the overlapped SVs between Ningxiang pig and Duroc pig,which had an impact on the overlapped region.The enrichment analysis indicated that these SV-associated genes were significantly enriched in pathways related to the Digestive system,Immune system,Nervous system,Lipid metabolism,and Environmental adaptation.In addition to a large number of SVs between the Ningxiang and Duroc genomes,the most remarkable difference between the two genomes was an inversion from 55.8 to 57.8 Mb on chromosome 6.The 49 genes were annotated in this region,which were involved in translation,signal transduction,inflammatory response,and lipid metabolic process.Meanwhile,the genes involved in lipid metabolism including ACOT13,PPARD,PPARG,PPARGC1 A,LPL,FADS2,FADS6 and SCD5 had significant SVs.The differences between Ningxiang pig,a fatty breed,and Droc pig,a lean breed,were analyzed by means of whole-genome structural variation detection.And then some dominant genes in the lipid metabolism of Ningxiang pig were explored.This study provided powerful tools and resources for the conservation of pig breeds and genetic resources.In order to further understand transcriptional regulation of lipid metabolism in different developmental stages,we chose four developmental stages including the piglet stage(30-day),the growing pig stage(90-day),the early fattening stage(150-day)and the late fattening stage(210-day).The piglets of four sows bred with the same boar were selected for this study,and three male offspring by the same sow were slaughtered for sample in each stage.The three piglets were full sibs at each time point while each three piglets were half sibs at different stages.The samples of longissimus dorsi muscle,back subcutaneous fa and liver,in twelve healthy offspring,were collected for transcriptome.The differential expression and function prediction of lncRNA and mRNA in four important developmental stages were analyzed by second generation high-throughput sequencing technology.STEM and WGCNA analyses were used to explore the possible targeted relationship between mRNA and lncRNA in development process.And some lncRNAs related to lipid metabolism were found in three tissues.Finally,RT-qPCR was used to the lncRNAs with high expression level and the mRNAs related to lipid metabolism,in order to verify the accuracy of high-throughput sequencing.(1)2683 novel lncRNA were identified in muscle tissue.Though STEM analysis,it was found that the lncRNA modules 15 and 16 had similar expression patterns with mRNA modules 15 and 16,respectively,and the target genes of lncRNA modules 15 and 16 were exactly in the corresponding mRNA modules,in which MSTRG.2011.1 and MSTRG.22358.1 targeted LEPR;MSTRG.19683.1,MSTRG.20242.2 and MSTRG.860.1targeted KLF7 and MSTRG.1058.1 targeted SGMS1.These regulatory relationships could be involved in lipid metabolism.Finally,the mRNA-lncRNA co-expression network was constructed by WGCNA.The hub gene PKD2 was associated with MSTRG.21592.2,MSTRG.8859.2 and MSTRG.18175.1 and the hub gene SOX13 was associated with MSTRG.21592.2 and MSTRG.9088.1.These associations could be involved in lipid metabolism.(2)2872 novel lncRNAs were identified in adipose tissue.By constructing closed and consecutive groups,there were targeted regulations between the common DElncRNAs and DEmRNAs,in which MSTRG.35604.2,MSTRG.2011.1,MSTRG.26481.9,MSTRG.23976.1,MSTRG.40648.1 and MSTRG.27163.1 targeted CYP2A29,AGMO,IGFALS,CES1,LEPR and PLAC8 involved in lipid metabolism.Though STEM analysis,it was found that the lncRNA modules 2 and 5 had similar expression patterns with mRNA modules 2 and 5,respectively,and the target genes of lncRNA modules 2 and 5 were exactly in the corresponding mRNA modules,in which MSTRG.18828.1 and MSTRG.15237 targeted ADCY9;MSTRG.1058.1 targeted SGMS1 and MSTRG.24004.1 targeted DECR1.These regulatory relationships could be involved in lipid metabolism.Finally,the mRNA-lncRNA co-expression network was constructed by WGCNA.,in which the hub gene PHACTR2,GPRC5 C and TMEM150 C could be involved in lipid metabolism.(3)2830 novel lncRNAs were identified in liver tissue.By constructing closed and consecutive groups,there were targeted regulations between the common DElncRNAs and DEmRNAs,in which MSTRG.33238.1 targeted CYP4A24 involved in lipid metabolism.Though STEM analysis,it was found that the lncRNA module 4 had similar expression patterns with mRNA module 4,and the target genes of lncRNA module 4 was exactly in the corresponding mRNA module,in which MSTRG.40598.2,MSTRG.31383.3,MSTRG.8684.1,MSTRG.33542.1,MSTRG.25050.1,MSTRG.18175.1,MSTRG.42785.1,MSTRG.42141.2,MSTRG.28249.2,MSTRG.13408.3 and MSTRG.28808.3 targeted CAV1 which could be involved in lipid metabolism.Finally,the mRNA-lncRNA co-expression network was constructed by WGCNA.The hub gene MED24 was associated with MSTRG.2158.2,MSTRG.34993.2,MSTRG.16183.1 and MSTRG.17517.2 and the hub gene ACADSB was associated with MSTRG.6256.1,MSTRG.22802.1,MSTRG11451.1,MSTRG.24004.1,MSTRG.26481.9,MSTRG.3283.1,MSTRG.29185.1 and MSTRG.3444.1.These associations could be involved in lipid metabolism.In this study,the whole genome data of Ningxiang pig was obtained by the latest third generation sequencing technology firstly.And then the whole transcriptome sequencing was used to the longissimus dorsi muscle,back subcutaneous adipose and liver of Ningxiang pigs by second-generation sequencing platform to study the regulation of genes related to lipid metabolism in development stages of Ningxiang pigs.This study will provide the molecular basis for the following research such as the candidate genes for the lncRNAs.Meanwhile,the developmental characteristics in muscle and adipose tissue of Ningxiang pigs can be learned in the actual production,which can provide a basis for the protection of germplasm resource and the selection of traits. |
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