Karyotype Analysis Of FB Hybrid Population,molecular Cloning Of Duplicated Fgf1 Genes In Grass Carp(Ctenopharyngodon Idella)and The Research Of HOX Gene Clusters In Crucian Carp(Carassius Auratus)

Blunt snout bream(Megalobrama amblycephala)and Topmouth culter(Culter alburnus Basilewsky)belongs to Cyprinidae,Megalobrama and Culter respectively,occupies an important place in the economic farmed fish in China.Through hybridization,we can obtained a newly hybrid cultivar synthesizes parents' eminent traits.There were significantly differences among hybrid offspring,analysising the karyotype of them is necessary to study the relationship between genetic composition and properties.The grass carp(Ctenopharyngodon idellus)is an important species for freshwater aquaculture,fast growth,individual big,provide an appropriate material for the growth research.Carassius auratus has the advantages of widespread,adaptable,high diversity,and genome duplication,is a good material for the study of genetics and evolution.Hox gene family evolved from the ancient ancestors gene,were highly conservative in the process of evolution.The structure and quantity of its parallel evolution of homologous genes were ever changing with the replication of the genome.All of this makes them become important model for the study on evolution and genome duplication.The study includes following three contents mainly:(1)In this study,we prepared the chromosome specimens of blunt snout bream(bsb),topmouth culter(tc),bsb(?)× tc(?)(FB),bsb(?)× FB(?),FB(?)× bsb(?),tc(?)× FB(?),FB(?)× tc(?),FB(?)× FB(?)by injecting PHA and colchicines.All of the hybrids possess 48 chromosomes.The karyotype of blunt snout bream is 2n=18m+26sm+4st,NF=92;The karyotype of topmouth culter is 2n=16m+26sm+6st,NF=90;The karyotype of FB is 2n=14m+28sm+6st,NF=90;The karyotype of bsb(?)× FB(?)is 2n=16m+28sm+4st,NF=92;The karyotype of FB(?)× bsb(?)is 2n=16m+30sm+2st,NF=94;The karyotype of tc(?)× FB(?)is 2n=12m+32sm+4st,NF=92;The karyotype of FB(?)× tc(?)is 2n=16m+30sm+2st,NF=94;The karyotype of FB(?)× FB(?)is 2n=14m+28sm+6st,NF=90.Obviously,the karyotype of hybrids was different with the expected results.The chromosomes of hybrids was not the simple combination of half of the parents chromosomes,but shows polymorphism.In addition,we measured the DNA contents of each hybrids by flow cytometry(BD Accuri? C6),explored the genetic composition of hybrids,all of this provides the reference basis for the study of fish crossbreeding.(2)Fibroblast growth factor 1(Fgf1)as a mitogenic factor,it is play an important role in the regulation of cell proliferation,growth and differentiation in vertebrates.In this study,we reported the isolation and expression characterization of two fgf1 genes in grass carp(Ctenopharyngodon idella).Grass carp fgf1 a and fgf1 b cDNAs are highly divergent,sharing a relatively low amino acid sequence identity of 50%,probably due to fish-specific gene duplication.fgf1 a and fgf1 b mRNAs were detected in the zygote and expressed throughout embryogenesis.Both fgf1 a and fgf1 b mRNAs were primarily detectable in the notochord at 12 hpf.At 24 hpf,fgf1 a mRNA was mainly expressed in the gut and somites,while fgf1 b transcript persisted in the notochord and was detected in the tailbud.At 36 hpf,both fgf1 a and fgf1 b transcripts were detected in the brain,somites,and tailbud.In addition,the fgf1 a mRNA was detected at the base of the yolk sac,whereas the fgf1 b mRNA was expressed in the pectoral fin.In adult fish,duplicated fgf1 a and fgf1 b mRNAs were distributed in most tissues.After 2-6 days of starvation,both fgf1 a and fgf1 b mRNAs were upregulated in the muscle and liver.In the brain,fgf1 a mRNA was upregulated,while fgf1 b mRNA was significantly downregulated at 6 days.Furthermore,both fgf1 a and fgf1 b mRNA levels were significantly decreased in the brain and muscle after administration of 10 or 50 ?g of the human growth hormone(hGH),while their mRNA levels were no significant difference in the liver.These results suggest that duplicated fgf1 s may play important but divergent roles in the grass carp development.(3)In this study,we screened 59 Hox amino acid sequences,including duplicated genes.Amino acid sequences were not all the same size,they are between 85 ~ 86 amino acids.All the Hox genes belongs to seven different gene cluster(HoxAa,HoxAb,HoxBa,HoxBb,HoxCa,HoxCb,HoxDa),including 6 HoxAa genes,10 HoxAb genes,10 HoxBa genes,8 HoxBb genes,14 HoxCa genes,3 HoxCb genes,8HoxDa genes.Each of the amino acid sequences has a conserved domain that contains about 60 amino acids.In the screened Hox genes,75% of them have two copies,and they were considered paralogous genes.The similarity of the amino acids coding by duplicated genes was between 44% ~ 99%.According to the composition of Hox genes,we speculate that the genome of the crucian carp happened “the fourth duplication”.Phylogenetic analysis showed that orthologous genes of the different species were clustered.There were four different Clustering types: crucian carp and common carp was the first cluster,this type was accounting for 41%,highlighted in red solid line;crucian carp and zebrafish was the first cluster,this type was accounting for 34%,highlighted in blue solid line;common carp and zebrafish was the first cluster which with crucian carp was the second cluster,this type was accounting for 12%,highlighted in black solid line;crucian carp and salmo salar or rainbow trout was the first cluster,this type was accounting for 13%,highlighted in yellow solid line.We can see crucian carp and other cyprinidae fish was clustered well,preliminarily concluded the genetic relationship.Base composition analysis showed that: the G-C content of duplicated genes were similar,but there are exceptions,such as A9b?/A9b?,B1a?/B1a?,B2a?/B2a?,B1b?/B1b?,B13a?/B13a?,C1a?/C1a?.On the one hand,we think that some of the duplicated genes were not contain all the CDS,it may be caused the G-C content differences.On the other hand,we think that the amino acid sequence similarity of duplicated genes may be caused the G-C content differences,when the amino acid sequence similarity of duplicated genes was low,its G-C content difference is bigger also.These results have laid a solid foundation for the further study about evolution and origin of crucian carp,also the study of calculate concrete time of the fourth duplication.