Identification Of Extra Chromosome Derive In The New Tetraploid Gibel Carp

Crucian carp is an important freshwater aquaculture species with high economic benefits,the main crucian carp species bred in China have Pengze crucian carp,Carassius auratus gibelio and other triploid crucian carp,only a few tetraploid individuals are naturally present.In our previously triploid allogynogenetic crucian carp bred practice,we found a gibel carp with significant growth advantage.The novel gibel carp strain with stable morphological traits were developed through systematic selection of six continuous generations,through DNA content analyzing and chromosome number counting of this selected strain demonstrating that they were tetraploids with 208 chromosomes(4n=208).However,how the tetraploidy generated and where the additional chromosomes derived from are not clear,whether the additional chromosomes were derived from chromosome doubling or alien chromosome integration require further investigations to confirm.In this study we using molecular biology to confirm where the additional chromosomes were derived from,the identification methods and the results are as follow:1.Microsatellite DNA(SSR),was identified as an ideal tracking markers in allele transmission.from parental to offspring.Microsatellite makers are highly polymorphic DNA markers.In the present study,98microsatellite makers isolated from the common carp and 20 microsatellite makers isolated from crucian carp,to detect triploid carp,crucian carp and carp new bred tetraploid gibel carp.We screened 9 microsatellite makers,the site which only in the new tetraploid gibel carp and carp have the same size bands.Some of these primers amplification bands was sequenced,by comparison,the results show specific fragments in tetraploid gibel carp and carp in almost the same,most of similarity of 99%and 100%.As a comparison,we sequenced the same size amplification bands which three fish all have in three microsatellite loci,the results also show that the similarity with tetraploid gibel carp is higher than with triploid carp.Thus,we considered that the additional chromosomes of the new tetraploid gibel carp were most likely derived from crap.2.Ribosomal DNA internal transcribed spacer(ITS),is often used to distinguish between different species and study the relationship between population,it is also a marker for germplasm identification and genetic diversity.In this study,the new breeding tetraploid carp ITS1,ITS2 was isolated in 20 individuals,and each sample sequenced 3 times in ITS1,ITS2 region.The results showed that ITS1 region amplified sequences in 20 sample of 60 sequenced have two types,the common carp type of 364bp and gibel carp type of 395bp;the gible carp type ITS1 was found only 5 sequences in 5 tetraploid individual,the probability of occurrence is 8.3%,the other 55 sequences were the commen carp type ITS1,the probability of occurrence is 91.7%.And the ITS2 region amplified of 60 sequences high identity in length,were the commen carp type ITS2 with 513bp.The analysis showed that tetraploid gibel carp was genetic mixture with the commen carp in the locus of ITS1,proved that the novel tetraploid gibel carp extra chromosome comes from carp.