Construction And Preliminary BAC-End Sequence Analysis Of A Bacterial Artificial Chromosome Library Of The Red Crucian Carp

Red crucian carp, with red body colour, belongs to Carassius, Cyprinidae, Cyp-rinidae, and is a variety of crucian carp. It has some excellent characters such as stable heredity characters, good viability, earlier sexual matuation and powerful reproductive capacity.The modified red crucian carp we used in the experiment is characterized by high dorsum and strong disease-resistance. Red crucian carp artificial Parthenogenetic Reproduction Technique is simple and well-established in our lab and is convenient to purify genes and build up inbred lines for different purposes quickly. All these advantages make the red crucian carp a good experimental animal model.What’s more, because red crucian carp is the maternal of the allotetraploid hybrid of red crucian carp(Carassiu sauratus red var.♀)×common carp(Cyprinus carpio L.♂), its genomic study, which hasn’t begun yet, is important for the evolution analysis of the allotetraploid hybrid. In order to better study its genome character and evolution, we construct the red crucian carp BAC library and sequence132red crucian carp BAC-end sequences(BES).3clones are selected to sequence the full-length. Results and conclusions are as follows.(1) The BAC library of red crucian carp contain14,850clones. The size of inserted fragments is between10kb and150kb, with80kb on average.(2) Using T7and M13primers, we sequence100randomly selected clones, and abtain132high quality BAC-Endsequences.The average length of these sequences is951bp, with37.5%GC, which is lower than that of channel catfish and medaka but slightly higher than zebra fish. Repeat sequence analysis by RepeatMasker indicate that DNA retrotransposons are predominant, followed by LINE and LTR, with no find of SINE.15simple sequence repeats (SSRs) are found by Trf, with10double-base repeats,5three or four-base repeats. These SSR markers would be very useful for further genetic linkage map construction and population genetics analysis.(3)On the basis of end sequence, we select three BAC clones to sequence the full-length which are labeled1,2and3, with total length50,982bp,15,323bp and9,663bp respectively. By GENESCAN analysis, No.1sequence contain7registered genes and two non-registered genes. The7registered genes are element Tf2155kDa protein type1-like, nanor b, similar to putative reverse transcriptase, putative reverse transcriptase, transmembrane protein196-like, hypothetical protein STHERM and ORF2-encoded protein. The average length is4515bp and promoters are forecasted in five of them. No.2sequence contain part of estrogen receptor beta gene,including5extrons and5introns sequence.The Phylogenetic analysis of carp, zebra fish, medaka and fugu partial coding region of estrogen receptor p gene reveal that red crucian carp and common carp share the highest homology. No.3sequence consist of36tandem repeats. Each repeat has263bp and is confirmed as red crucian carp special probe through FISH. This repeat sequence could be used to explain the composition of allotetraploid hybrid and its chromosome exchange and recombination during meiosis.