Studies On The Heredity Characteristic Of White Crucian Carp(Carassius Auratus Cuvieri)(♀)X Red Crucian Carp(Carassius Auratus Red Var.)(♂)and Its Genetic Modification

Crucian carp is one of the most popular freshwater fishes in China.Due to its tender and delicious meat and moderate size,it has been in short supply for a long time.Hybridization is an effective breeding technique and is widely used in fish.In this study,we obtained the hybrid lineage(WR F1-F4)derived from the intraspecific hybridization of white crucian carp(Carassius auratus cuvieri,WCC,♀)and red crucian carp(Carassius auratus red var.,RCC,♂).The genetic characteristics and biological characteristics were studied in our research.The major results were presented as following:1.Measurable and countable traits were examined in each sample of WR-F1-WR-F4.The results showed all the assessed morphological data were not significant between the hybrids,suggesting the hybrid progenies are highly stable.The ploidy and chromosome composition of WR-F1-WR-F4 were confirmed by counting chromosomal number and measuring DNA content.The results showed all the progenies are diploid and have 100 chromosomes.Histological sectioning was used to examine gonad development in WR-F1-WR-F4.The results showed the ovaries developed normally and were mainly composed of oocytesat phaseⅣ-stage;and the testes of the hybrids were full of mature sperm.The hybrids were sexually mature at one-year-old.White sperm or mature ova could be stripped out from 12-month-old hybrids.In addition,they exhibited high fertilization rate(90.2%-91.3%)and high hatchery rate(81.5%-82.7%),which contributed to produce and enlarge the population.At molecular level,the sequencesof 5S rDNA showed high similarity with its parents and showed some mutants which can inherit to the next generation steadily.Different from parents(silver gray and red),all the hybrids with the gray color showed the hybrid morphologicaltraits of both parents.We obtained a new type fish derived from the hybridization of female WR-F1 and male WCC;and a new type triploid fish derived from the hybridization of female WR-F1 and male allotetraploid fish.2.The nutritional composition and quality of muscles from WR and its parents were investigated and analyzed by biochemical analysis methods.The results showed that the moisture content of WR(71.00%)was significantly lower than that of its parents(WCC:75.60%,RCC:75.50%),while the protein content of WR(17.70%)was higher than that of RCC(17.00%)and was much higher than that of WCC(14.80%).Fifteen kinds of amino acids were found in muscles from WR and its parents.Both total amino acids and total essential amino acids content of WR(15.87%and 6.55%)were higher than those of RCC(15.52%and 6.64%respectively),and were significantly higher than those of WCC(13.13%and 5.27%respectively).In particular,the total flavored amino acid content of WR(6.26%)was higher than that of RCC(6.07%),and was much higher than that of WCC(5.29%).3.Under the same breeding conditions,the average weight of 12-month-old WR was over 350 g,which was close to the growth rate of WCC(319 g)and significantly faster than RCC(265 g)(p<0.05).Transcription of GH,GHR and IGF-1 mRNA were quantified by real-time PCR.The results showed that there was no significant difference of the transcription of tested genes between WR and WCC,but there was significant difference between WR and RCC(p<0.05),suggesting WR inherit the growth rate of WCC.4.The entire mitochondrial DNA(mtDNA)of WR and its parents was sequenced and analyzed.The complete mtDNA sequence of WR was 16580 bp in length,the same as WCC and RCC.Using a variety of bioinformatics software,we analyzed mtDNA structures,gene order,and sequence similarity comparison.The results indicated that the complete mtDNA of WR was quite similar to that of WCC(99.22%)rather than RCC(98.63%),suggesting WR was still strictly maternal inherited.5.We presented an analysis of the transcriptomes from the livers of WCC,RCC,WR-F1,and WR-F2 to reveal the genomic change of the progenies.We compared the orthologous genes in the liver transcriptomes of hybrids with those of WCC and RCC,and classified the orthologous genes into eight gene patterns within three categories(chimera,mutant,and biparentalorigin genes).The results revealed 19.04%,4.17%chimeric genes and 6.90%,5.05%mutations of orthologous genes in WR-F1 and WR-F2 respectively.Seventeen of twenty-three characterized genes(77%)were confirmed to be the chimeras at the genomic DNA level.The functional analysis used by KEGG and GO classification discovered that some chimeric and mutant genes were related to metabolic process,immune system and developmental process in WR-Fi.6.In addition,we also conducted some studies on genetic engineering breeding for Crucian carp.Using the method of gene knockout,it can not only study the function of the related genes,but also improve the genetic improvement of Crucian carp.Gene knockout technique provides a safe and acceptant way for fish breeding.Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic improvement and hybridization breeding.In order to explore the application of gene knockout technique in hybrid fish,we took advantage of the CRISPR-Cas9 system to knockout the tyrosinase(TYR)in WCC and WR.Both the WCC and WR mutant showed different degrees of melanin reduction compared with the wild-type sibling control fish,resulting from different mutation efficiency ranging from 60%to 90%.We confirmed the results by measuring the tyr mRNA transcription and TYR expression.In addition,the transcriptional profiles of a series of pivotal pigment synthesis genes were down-regulated in tyr-CRISPR WCC.Taken together,these results demonstrate that the CRISPR-Cas9 system is an effective genome-editing tool for WCC and its hybrids for studying genetic improvement and breeding.Furthermore,the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formationin non-model fish.