| In China,Cyprinus carpio var.singuonensis and Carassius auratus var.pengzesis were important freshwater aquatic animals.The previous study had obtained allotetraploid Pengze crucian carp through gynogenesis induced by the sperm of Cyprinus carpio var.singuonensis.In oeder to explore the difference between the allotetraploid Pengze crucian carp and female parent Pengze crucian carp,the fertilization data,embryonic development and growth-related gene expression were studied and compared.Addtionally,a pair of molecular markers that could rapidly identify the two fish were developed.The main findings are as follows:1.Embryonic development of allotetraploid Pengze crucian carp and Pengze crucian carpUsing the sperm of Xingguo red carp to induce the gynogenesis of allotetraploid Pengze crucian carp and Pengze crucian carp resulted in a high fertilization rate,hatching rate and seedling rate.We discovered that the fertilization rate,hatching rate,deformity rate and emergence rate of allotetraploid Pengze crucian carp were subtantially higher than Pengze crucian carp.Inadditon,the fertilized eggs of both fishes were spherical or oval viscous eggs with light yellow evenly distributed egg yolk.The incubation time of allotetraploid Pengze crucian carp and Pengze crucian carp fertilized eggs was 57 h 11 min and 66 h 29 min,respectively,at the water temperature of 24±1℃.The embryonic development process and the characteristics in each stage of the two fish were basically the same,which had gone through a total of 26 stages,including fertilization,cleavage,blastocyst,gastrulation,neurite,organ differentiation,and membrane emergence.The statistical analysis revealed that the diameter of fertilized eggs of allotetraploid Pengze crucian carp(1.73±0.01 mm)was significantly larger than that of Pengze crucian carp(1.66±0.017 mm),and the length(5.18±0.17 mm)of newly hatched larvae of allotetraploid Pengze crucian carp was significantly longer than that of Pengze crucian carp(4.78±0.07mm);allotetraploid Pengze crucian carp embryos(1372.6 h·℃)had a lower accumulated temperature of embryonic development than Pengze crucian carp embryos(1595.6 h·℃).The results revealed that both fish have normal reproductive ability through heterosperm insemination,and allotetraploid Pengze crucian carp’s reproductive performance is better than that of Pengze crucian carp,although the deformity rate is higher.Secondly,above indicated that the allotetraploid Pengze crucian carp embryos developed normally,and the embryos developed faster than that of the maternal Pengze crucian carp,and the accumulated temperature required for embryonic development was lower than that of the maternal Pengze crucian carp.2.The expression analysis of allotetraploid Pengze crucian carp and Pengze crucian carp growth-related genesThe allotetraploid Pengze crucian carp’s body length(141.43±7.27 mm)and body weight(88.6 ± 13.6 g)at 12 months old were substantially greater than Pengze crucian carp’s(129.34±8.03 mm)and body weight(61.53 g ± 12.6 g).The results showed that the growth rate of allotetraploid Pengze crucian carp was faster than that of Pengze crucian carp.The cDNA sequences of IGF-2,IGF-1 and GH of allotetraploid Peng Ze crucian carp and Pengze crucian carp were cloned by RT-PCR based on the cDNA sequences of GH,IGF-1 and IGF-2 of carassius auratus gibelio,Pengze crucian carp and goldfish.The GH,IGF-1,and IGF-2 cDNAs lengths of allotetraploid Pengze crucian carp were 992 bp,685 bp,and 782 bp,respectively;while the GH,IGF-1,and IGF-2 cDNAs lengths of Pengze crucian carp were 993 bp,644 bp and 780 bp,respectively.The open reading frames for GH,IGF-1 and IGF-2 in the two fishes were 633 bp,486 bp and 605 bp,respectively.Furthermore,the amino acid sequences of GH,IGF-1,and IGF-2 of the two fish were identical.In addition to homology analysis of GH,IGF-1,IGF-2 with other species was carried out by BLAST software.The homology of GH gene amino acid sequence were99.52%(Carassius auratus),99.05%(Carassius auratus gibelio)and 96.19%(Cyprinus),respectively.The amino acid sequence homology of IGF-1 gene was 100%(Carassius auratus)and 96.27%(Cyprinus),respectively.The amino acid sequence homology of IGF-2 gene was 99.5%(Carassius auratus)and 90.55%(Cyprinus),respectively.The GH,IGF-1 and IGF-2 genes of the two fishes were highly homologous with those of crucian carp and Cyprinus.When expression levels of GH,IGF-1,and IGF-2 were measured in the brain,pituitary,liver,and muscle tissue of the two fish,it was discovered that the GH,IGF-1,and IGF-2were all expressed in the four tissues.The pituitary gland has the highest expression of GH and lower expression in other tissues,The specific expression levels of each tissue are ranked from high to low: pituitary>brain>liver>muscle;IGF-1 is mostly expressed in the liver,with the highest level of expression,with lower levels of expression in other tissues ranging from high to low: liver>muscle>brain>pituitary;IGF-2 is mainly expressed in the liver,and the order of expression in various tissues from highest to lowest,is as follow:liver>muscle>pituitary>brain.In additon,the expression of GH in the pituitary of allotetraploid Pengze crucian carp was significantly higher than Pengze crucian carp,and there was no significant difference in the expression in brain,iver,and muscle tissue.The expression level of IGF-1 in allotetraploid Pengze crucian carp liver group was significantly higher than Pengze crucian carp,whereas the expression level of IGF-1 in allotetraploid Pengze crucian carp muscle tissue was higher than Pengze crucian carp,with no significant difference in the expression levels in pituitary tissue and brain tissue.The expression level of IGF-2 in allotetraploid Pengze crucian carp’s liver and brain was significantly higher than in Pengze crucian carp,but there was no significant difference in muscle or pituitary gland tissue.These results indicate that the pituitary of the allotetraploid Pengze crucian carp and Pengze crucian carp is the site of GH synthesis,and the liver is the synthesis site of IGF-1 and IGF-2.In addition,the rapid growth rate of the allotetraploid Pengze crucian carp Growth may be related to the high expression of GH,IGF-1 and IGF-2.3.Molecular marker development of allotetraploid Pengze crucian carp and Pengze crucian carpThe specific fragments of carp were obtained by comparing the gene sequences based on the genome data of carp and crucian carp on NCBI and a specific molecular marker with a single band of 1000 bp that could be amplified in allotetraploid Pengze crucian carp but not in Pengze crucian carp was obtained.This molecular marker was proved to be durable and efficient in identifying allotetraploid Pengze crucian carp and Pengze crucian carp,laying the foundation for future allotetraploid Pengze crucian carp breeding research. |
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