| The proposal of marker-assisted breeding breaks the traditional concept and provides new idea and shortcut for animal genetic breeding.It is also an important way to shorten the breeding cycle and quickly cultivate excellent variety of Pelodiscus sinensis.However,molecular genetic basis of this species is weak,and the reliable molecular markers are indistinct,which affects the development of functional genes and the progress of improved breeding.This study was intended to develop and identify genes and SNPs associated with growth trait based on whole-genome resequencing and transcriptome sequencing.These results provided valuable genetics resources for marker-assisted breeding and dissected the genetic mechanism of the growth trait in P.sinensis.The main progress made in this study is as follows:1.Transcriptome analysis and identification of genes associated with growth traits in P.sinensisTo identify growth-related genes and explore the molecular mechanism of its growth regulation,fast-growing and slow-growing turtles were selected from two growth stages,juveniles or adults,respectively,for transcriptome sequencing using liver tissue on Illumina platform.Analyses showed that,303 and 1093 genes were differentially expressed in juveniles and adults,respectively.KEGG enrichment analysis of DEGs in juveniles identified only one pathway being significantly enriched,the PPAR signaling pathway,while eight pathways mostly related to fatty acid metabolism were identified in adults.These findings suggest that regulation of growth is more complicated and involves more genes and pathways in adults compared with juveniles,and metabolic or metabolic-related genes may relate to the growth difference of P.sinensis.Some key genes related to growth were identified from growth-related signaling pathways,and GH-IGF 1 axis genes might play important roles in the growth difference of P.sinensis.2.Development of SNP based on P.sinensis transcriptome and association analysis with growth traitsA total of 74,100 putative SNPs were predicted,located in 12,417 expressed genes,which would enrich the basic database of P.sinensis.Among which,3,142 putative SNPs were annotated to 429 DEGs.From these putative SNPs,a total of 108 SNPs were verified from 17 DEGs,and 58 SNPs were genotyped successfully using tetra-primer amplification refractory mutation system PCR.Growth association analysis revealed that a missense mutation in SLC27A2 is associated with growth traits,suggesting this gene is a good candidate for further growth studies in P.sinensis.3.Sequence analysis,homology comparison and tissue distribution of IGFsThe c DNA sequences and encoded amino acids of 10 IGFs system gene were analyzed in P.sinensis.Compared to other vertebrates,the IGF ligands(IGF1 and IGF2),receptors(IGF1R and IGF2R)and part of IGFBPs(IGFBP3 and IGFBP4)were highly conservative,while the N-terminal domains of IGFBP2,IGFBP5,IGFBP6 and IGFBP7 were missing,suggesting their function in P.sinensis might be different from other species.To expand the understanding of these genes,qualitative expression of IGFs system genes across tissues and development stages were performed.The results showed that,the expression of IGFs system genes was ubiquitous across tissues(liver,hypophysis,lung,muscle,spleen,kidney,heart,gonad,intestine)in adults,apart from IGFBP1,which was variable.It was found that the m RNA expression levels of IGF1,IGF2,IGFBP2 and IGFBP4 genes were positively correlated with the rapid growth of individuals,and the high expression levels of IGF2 R and IGFBP1 gene were related with the slow growth of individuals.4.SNP detection of IGFs and its association with growth traits of P.sinensisSeventy-five SNPs were detected from the 3’UTR,5’UTR,exons,and some intron regions adjacent to the exons of IGFs system genes using combining direct sequencing with multiple sequence alignment.Fifteen SNPs were genotyped successfully.Growth association analysis revealed that one synonymous mutation in the eighth exon of the IGF2 R gene and one mutation in the 3’UTR of the IGFBP1 gene were significantly related to growth traits,suggesting that these two genes might play important roles in the growth regulation of the P.sinensis.5.Location and mapping of the growth-related genes of P.sinensis using whole-genome resequencingBy whole-genome resequencing,a total of 18,920,618 SNPs and 3,287,872 In Dels were detected.The bulked segregation analysis was used to achieve the preliminary mapping of the underlying growth-associated genes.Ten significantly related scaffolds were identified in the female,among which 22 genes containing non-synonymous mutation were annotated,including 6 trypsin homologous genes and 5 butyrophilin subfamily genes,suggesting that genes related to digestion and immune may be the key genes in growth regulation of female P.sinensis.Four scaffolds that were significantly related to the target trait were identified in the male,and four genes containing non-synonymous mutation were annotated.Three of them belonged to the butyrophilin subfamily,indicating that immune-related genes may also affect the growth performance of male P.sinensis. |
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