Study On Ploidy Identification, Morphology, Growth Performance And 5SrDNA Sequence Of A New Tetraploid Carassius Auratus

Carassius auratus var.pengsenensis was produced in Pengze County,Jiujiang City,Jiangxi Province,which belongs to the genus Carassius of Cyprinidae,Cypriniformes.Because of its characteristics of fast growth,large size,strong disease and stress resistance and rich nutrition,it become an excellent variety for freshwater aquaculture.In previous study,a few tetraploids(3 females,2 males,4N=200+)were obtained by gynogenesis through heterogeneous sperm stimulation based on Cyprinus carpio var.singuonensis(2N=100)and Carassius auratus var.pengsenensis(3N=150+).Then we reproduced to get a second generation.In this study,the ploidy identification,morphology,growth performance and 5SrDNA sequence analysis of the second generation of Carassius auratus var.pengsenensis were carried out.The results will provide basic data for the cultivation of a new tetraploid Carassius auratus.The main research contents are as follows.1 Ploidy identification and chromosome karyotype analysis of offspring of allotetraploid Carassius auratus var.pengsenensis.In this study,using Carassius auratus var.pengsenensis as the internal reference,the relative DNA content was measured by flow cytometry;The red blood cell morphology and size of Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis were compared;Offspring of allotetraploid Carassius auratus var.pengsenensis muscle and fin tissue was taken foe cell culture,and chromosome samples were prepared for chromosome count and karyotype analysis.The results showed that:Offspring of allotetraploid Carassius auratus var.pengsenensis was still tetraploid;The long axis,surface area,volume,long axis,surface area and volume of red blood cells of offspring of allotetraploid Carassius auratus var.pengsenensis were significantly larger than those of Carassius auratus var.pengsenensis;From the morphological point of view,the red blood cells of offspring of allotetraploid Carassius auratus var.pengsenensis were longer than those of Carassius auratus var.pengsenensis;The karyotype formula is 4N=42m+64sm+62st+32t,NF=368.The extra chromosomes are supernumerary chromosomes and no atypical sex chromosomes were found.Offspring of allotetraploid Carassius auratus var.pengsenensis belongs to the median evolution group.2 Study on morphological of offspring of allotetraploid Carassius auratus var.pengsenensis.In this study,the morphometric characters,meristic characters and frame structure characters of Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis were collected by X-ray photographic method.Multivariate statistical analysis was then performed,including independent sample T test,principal component analysis,discriminant analysis and difference coefficient analysis.The results indicated that Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis had four dorsal fin spines,three anal fin spines,four predorsal vertebrae,and two swim bladder sacs.However,it is worth noting that we found a significant difference in the numbers of caudal fin rays between Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis,which can be a distinguishing trait.Although there were significant differences found in other characteristics,the coefficient of difference(CD)remained below the subspecies level(1.28).Summarising these differences would have required the use of several independent factors,which would have been difficult.3 Study on growth performance of offspring of allotetraploid Carassius auratus var.pengsenensis.Through the 180 d breeding,parallel group weight and body length data were taken at 0,30,60,90,120,150 and 180 d.And then the growth performance of Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis were analyzed.The results show that:during the 180 d experiment,the body weight and body length of offspring of allotetraploid Carassius auratus var.pengsenensis were significantly different from those of Carassius auratus var.pengsenensis;At the end of the experiment,the body weight and body length of offspring of allotetraploid Carassius auratus var.pengsenensis were 30.18%and 7.70%higher than those of Carassius auratus var.pengsenensis;The absolute weight gain rates of Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis were different in different growth stages,and decreased to the minimum at 90～120 days.The absolute growth rate also showed a changing trend.At 90～120 days,the absolute growth rate of Carassius auratus var.pengsenensis was higher than that of offspring of allotetraploid Carassius auratus var.pengsenensis;Comparing the accumulation volume of Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis,it was found that the accumulation volume of them was different except for 90～150 d(P<0.05),and it reached the maximum value at 30～60 d,which was 0.0105±0.0012;During 30～180 d,the coefficient of fatness of offspring of allotetraploid Carassius auratus var.pengsenensis was significantly different from that of Carassius auratus var.pengsenensis,indicating that offspring of allotetraploid Carassius auratus var.pengsenensis were more obese;And then we fit the relationship between body weight and body length in different periods of Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis to get the growth curve.offspring of allotetraploid Carassius auratus var.pengsenensis:W=0.032L2.9915,R2=0.9779;Carassius auratus var.pengsenensis:W=0.0307L2.9941,R2=0.9827.All of the curve fit is good.To sum up,offspring of allotetraploid Carassius auratus var.pengsenensis show good growth performance.4 Study on 5SrDNA sequence of offspring of allotetraploid Carassius auratus var.pengsenensis.To find the molecular markers for identifying parents and offspring,seven pairs of primers were designed and synthesized for PCR amplification and cloning sequencing of 5SrDNA sequences of Cyprinus carpiouar var.singuonensis,Carassius auratus var.pengsenensis and offspring of allotetraploid Carassius auratus var.pengsenensis,and the sequencing results were analyzed by BLASTn and homology comparison.5 SrDNA molecular sequence analysis indicated that Cyprinus carpiouar var.singuonensis had two 5SrDNA structures(203 bp and 406 bp),of which 406 bp bands were two 203 bp repeats;Carassius auratus var.pengsenensis has three 5SrDNA structures(203 bp,304 bp,459 bp);Although some base mutations and losses occurred,offspring of allotetraploid Carassius auratus var.pengsenensis retained the 5SrDNA structure and organization characteristics of the parent(203 bp,406 bp sequences of Cyprinus carpiouar var.singuonensis and the 304 bp,459 bp sequences of Carassius auratus var.pengsenensis).Importantly,the results proved that the parents have crossed.