BAC Library Construction And Genetic Variation Of Functional Genesin Allotetraploid Hybrids

Allotetraploid hybrids (4nAT)were devrived from the interspecific hybridization from red crucian carp (carassius auratus red var.,♀,2n=100) xcommon carp (cyprinus carpio L.,(?),2n=100). In this hybrid lineage, the F1and F2were fertile diploid hybrids. The F3generation produced allotetraploid fertile individuals of both sexes. The bisexual fertility character maintained the tetraploidy from one generation to another. Currently, this allotetraploid hybrid lineage has been bred up to the23rd generation, which formed an allotetraploid stock (F3-F23). The genetic lineage, including the parents and each hybrid generation is clear. Allotetraploid hybrid was the first reported artificially cμltured allopolyploid with bisexual fertility and stable inheritance in fish which has played an important role in both aqucμlture and scientific study. In aquacμlture, large amount of cμltured tripolyploid fish were produced through the hybridization of red crucian carp(♀) x4nAT ((?)) which has great economic benefit in Chinese fishery and improved the progress of the selection breeding of fish. While forthe scientific study, as allotetraploid hybridisthe progeny of red crucian carp (♀) xcommon carp ((?)), it inherited the genetic material from both parents, thechromosome number has changed from100to200, in addition, allotetraploid hybrids haveshown some genetic variationcompared with their original parents in the genetics, cytology, evolutionary biology and other study which make allotetraploid hybridshave the potential to become a new species in future. To do research on the genome of allotetraploid hybridswill allow us to investigate the mechanism of interspecific hybridization, the genome evolution, the fate of allopolyploid animal hybrids and other essential biology phenomena.In this study, based on the BAC library of allotetraploid hybrids,14BAC clones were randomly chosen and the whole length was sequenced by ABI3730sequencer. Preminilary analysis of the genome characteristic of allotetraploid hybrid (include GC content, the proportion of repetitive sequence, etc.)and intron evolution through comparative study have been conducted.In addition,7orthologous gene of red crucian carp, common carp and4nAT were amplified, comparative study have been performedon exon, intron and whole DNA sequence level.1. BAC library construction of allotetraploid hybridsThe BAC library of allotetraploid hybrids was construced in our lab, in total,31,023positive clones werepicked up, after NotI digestion detection, the mean size of BAC clone was45.6kb, and all the clones were preserved in96-well plate in-80℃with a total number of324. Compared to other BAC libraries constructed from other species, the mean size of our BAC clone is shorter than others’also the whole library coverge is low, which may due to the complicated genome structure of allotetraploid hybrids. Although our BAC library is not complete and there are some shortage, however, for now, on the condition that the genome sequence data of allotetraploid hybrids is lack, the BAC library will provide great resources for scientific research of allotetraploid hybrids, it will lay foundation on whole genome sequencing project in later period, meantime it will provide precise resource for functional genomics and compartive genome study between allotetraploid hybrids and its original parents.2. BAC clone whole length sequenceTo preminilary investigate the genetic characteristic and proportion of repetitive sequence in allotetraploid hybrids,14randomly selected BAC clone were chosen for whole length sequencing, the sequence length of14BAC clone was ranged from17,849bp to87,725bp (without7.8kb vector sequence). The GC contentranged from34.38%to39.80%with the mean GC content is37.10%, while the proportion is a little higher than its male parent common carp (36.8%), higher GC content may caused by its higer level of methylation. In total,14BAC clone whole length sequence generated591,126bp which covers0.02%of4nAT genome (3.78Gb). Through RepeatMasker software,103,411bp (17.46%) repetitive sequences were obtained.The most abundant type of repetitive elements in the allotetraploid hybrids genome was retroelements (6.47%), including LINEs (2.72%) and LTR elements (3.66%) followed by DNA transposons (4.17%), other repeats, like105small RNA repeat (1.59%), were also identified.In common carp, the proportion of DNA transposons was6.67%while the proportion of retroelements was4.52%, it showed great difference comparing with allotetraploid hybrids.As in allotetraploid hybrids,Lower proportion of DNA transposons and higher proportion of retroelementswere detected compared with common carp.Allotetraploid hybrids were hybrid progeny, quite a number of genetic recombination, gene rearrangement and gene redundancy events will occur in this hybrid lingeage, new retroelement inserted into the new doubled genome will break the genome structure, lead to some gene silent events in order to regμlate the functional gene expressionwhich may elucidate the higher proportion of retroelement in allotetraploid hybrids.3. Intron evolution in allotetraploid hybridsThrough the FGENESHsoftware, we obationed11completely gene from14BAC clone whole length sequence, among them9orthologous genes were found in other model fish species and fish with whole genome data, after analyzing the gene structure of above species, we separatedthe exon and intron sequence, the detailed intron information (intron numbers, intron length, etc.) from4nAT, zebrafish, medaka, fugu, tetraodon, tilapia,lamprey, cod, stickleback,xenopus, and human was recorded and analyzed through Malin software, the resμlts indicated that the intron of4nAT was in dynamic process with30gained introns and39lost introns, which indicated rapid intron evolution in allotetraploid hybrids compared to other studied fish species.Based on the whole orthologous gene sequencesof allotetraploid hybrids and other studied fish species, the intron number and intron length of all studied species were obtained. In all studied teleost fishes, the mean intron length of zebrafish was32,164bp which is the longest, while the mean intron length of4nAT was shorter than zebrafish’s, lamprey’s and cod’s but longer than other teleost fish species. The mean intron density was among9.4to16per gene, while lamprey has the highest intron density which is16, however,4nAT has the lowest intron density which is9.4. From all the resμlts,4nAT showedgreat variation, the intron variation related to many factors, include generation period, gene expression, intron phase and so on, while in our resμlts we predictedthat interspecific hybridization may another key factors for intron evolution, which indicated interspecific hybridization of red crucian carp xcommon carp was the primary cause for the rapid intron evolution inallotetraploid hybrids.4. Comparative study among allotetraploid hybrids and its orginal parentsConserved primers based on BAC clone sequence and gene sequence of zebrafish were designed in order to amplify the gene sequences of4nAT, red crucian carp, and common carp.Using these primers,7homologous partial or whole gene sequence of4nAT, red crucian carp, and common carp were obtained. From exon, intron and whole DNA sequences level, phylogenetic tree was constructed by MEGA5which indicated that4nAT not only inhereit the genetic characterstics from its original parents but also show some genomic variation. On the whole DNA sequences level, through the mμltiplealignment of4nAT and its parents by using Clustal W software, there are two patterns of genomic DNA in the4nAT lineage including "genetic inheritance pattern" and "genetic variation pattern". In detail, eight subcategores coμld be classified. Among the gene sequence in4nAT, importin-13-like gene and fizzy-related protein homolog gene sequence showed high sequence similarity compared with their parents coμld be defined as "genetic inheritance pattern" which indicate that4nAT inherit the genetic materials from its parents. In addition, like denticleless homolog, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-5’, chemokine (C-X-C motif) receptor7b and phosphodiesterase11A gene, homoeologous recombination were detected in4nAT, meantime, iqcalwere confirmed to be a novel gene in 4nAT which showed quite different gene sequence and gene structure from its parents, all these gene sequences were defined as "genetic variation pattern" which indicate lots of genomic DNA changes have occurred in4nAT.In conclusion, based on the preliminary genome characteristic of allotetraploid hybrids, the intron evolution analysis and comparative study resμlts, we woμld like to indicate that allotetraploid hybrid is in rapid evolution since its formation.Our resμlts strongly suggest that interspecific hybridization is the primary reason for the genome changes of allotetraploid.