| A bisexual fertile and stable inheritabl the allotetraploid (AT) crucian-carp population was obtained originally by crossing red crucian carp(Carassius auratus red var., ,2n=100) with common carp (Cyprinus carpio L., S,2n=100). Up to date,18th generation of the allotetraploid crucian-carp was produced and became a new large allotetraploid folk. The formation of these folk has a great significance in understanding of fish evolution and genetic breeding. Studies indicated that there are lot of changes in genotype and phenotype between Allotetraploid crucian-carp and their diploid parents. Thus, it is essential to make deep systematic studies on its characterization in genome and transcriptome. For futher study of the Allotetraploid crucian-carp genomics, it is important to identify functional genes and biomarker relative to genetic traits and genetic mapping. It is benefical for us to establish a platform for molecular breeding.Here we constructed BAC (Bacterial artificial chromosome) library of Fig Allotetraploid crucian-carp, sequenced transcriptome of the allotetraploid crucian-carp liver, red crucian carp liver and common carp liver using 454 GS FLX,analyzed bioinformaticaly the data produced by 454 GS FLX. The major results were presented as following:1?For the first time we constructed a highly redundant genomic BAC library using high molecular weight(HMW) DNA from the allotetraploid crucian-carp produced in our fish development bioloy Lab by crossing red crucian carp(Carassius auratus red var.,9,2n=100) with common carp(Cyprinus carpio L.,,2n=100). the BAC library we constructed consists of 43200 clones in total. High quality genomic HMW DNA extracted from blood cells embedded in low-melting-point agarose plug was partially digested with Hind III,150-300kb fragments were isolated by pulsed field gel electrophoresis (PFGE), then 150-300kb fragment were purified with dialysis.ligated to linearized and phosphorylased vector pCClBAC, transformed into E.coli.the constructed BAC library with the insert fragments size about 110-310kb,the average size around 190kb as estimated by PFGE. The representation of the constructed BAC library is about 2.6 equivalents to the Fig Allotetraploid crucian-carp genome. All these BAC clones arepreserved in 450 pieces of 96-well plates in-80?refrigerator. The se reusts show that the BAC library will be usefull for identifying functional genes, genetic mapping, sequencing of the Allotetraploid crucian-carp genome, evolutional molecular machnism and trangenetic study.2?The transcriptomic raw data of the Allotetraploid crucian-carp liver, red crucian carp liver and common carp liver produced by 454 GS FLX are respectively about 1.2Gb,934Mb and 1.1Gb. After basical analysis,9554 Unigene were obtained.3?By using EDEGR, differential expression was analysized between Fi8 and red crucian carp. The result showed that 6192 contigs were upregulated and 217 contigs were downgulated (cutoff:logFC> 2, P< 0.05). Compared with common carp,4867 contigs were upregulated and 578 contigs were downregulated (cutoff:logFC> 2, P<0.05)4?Based on KEGG pathway analysis, the differential expression genes were identified. The result showed that the upregulated genes in F18 were mainly in Carbohydrate Metabolism, Amino Acid Metabolism, Lipid Metabolism and Energy Metabolism. Go analysis indicated that in molecular function level differential genes are mainly in binding and catalytic activity. Cytoplasm and organelle are the most of celluar component, while metabolic process and regulation of biological process contribut the most part of biological process.5?F18 contigs were mapped with red crucian carp and common carp reads. The results showed that 185 Unigenes of F18 contains fragments of red crucian carp, while 192 Unigenes of F18 contains fragments of common carp.24 Unigenes contains fragments from both red crucian carp and common carp.6?Between F18 and red crucian carp,46449 SNP sites were found, while 59436 SNP sites were found between F18 and common carp. Compared with red crucian carp, more SNP sites observed in common carp. This result proved that partial maternal inheritance of F18 in genetic characteristics. Transferrin variant A and MHC class I antigen had the most SNP sites, which potentially due to its increased resilience.7?Evolution analyis of transferrin variant A and MHC class I antigen:Phylogenetic tree of transferrin variant A showed, the sequence of F18 was more similar to red crucian carp compared with common carp While, MHC class I antigen of F18 showed high similarity with, common carp. This result suggestted that in F18 was in inherit both genetic information from parents and different genes show different deflection. From molecular level to decipher ploidy evolution process, all the genes face selection pressure of evolutionFor further study of molecular breeding and identifying functional genes, F18 BAC library was constructed?In addition, using 454 high throughout sequence technology, F18 and its parents liver transcripton were sequenced and then analysized using bioimformatics tools. The structure and differential expression of genes were studied. With the genes functional annotation, we offered more information for understanding ploidy evolution of fishes and molecular breeding... |
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