Establishment Of Improved Gynogenetic Grass Carp Population And Its Genetic Characterization

Grass carp(Ctenopharyngodon idellus,GC,2n=48)was an important economic fish with the highest production in China.According to fisheries yearbook of 2019,the production of GC reached 5.5 million tons in 2018.However,continuous manual self-crossing had led to their germplasm degradation,which further resulted in their disease and death.In order to produce improved GC,for the first time,the ultravioletirradiated sperm of koi carp(Cyprinus carpio haematopterus,KOC,2n =100)was used as a source of sperm stimulation to produce a new type of gynogenetic grass carp(GGC).In this study,we had established GGC line and conducted a systematic study of their genetic and other biological characteristics.1.For the first time,GGC had been produced by using ultravioletirradiated sperm of KOC to activate eggs of GC,following the treatment of doubling the chromosomes.In this way,a line of GGC had been established with 1310 individuals,which provided valuable experimental materials for the study of their genetic and other biological characteristics,and lay an important germplasm resource for their application in production.2.In this study,the basic morphological characteristics,red blood cell characteristics,chromosome number,and DNA content of GGC had been systematically studied.The results showed that GGC had highsimilarity characteristics with GC in body color,beard and body shape;no significant difference between GGC and GC in volume of red blood cells and DNA content(P>0.05),which indicated they had the same genetic material;GGC(2n=48)had 48 chromosomes with karyotype of18m+24sm+6st,which was the same to GC(2n=48),indicating that GGC was diploid;In summary,GGC and GC had similar biological characteristics.3.A systematic study on the molecular genetic characteristics of GGC.(1)The microsatellite DNA analysis was used to find a specific microsatellite DNA fragment(MFW1-gynogenetic grass carp,MFW1-G)derived from the paternal parent KOC in GGC.MFW1-G could be amplified in GGC and KOC but not in GC.For the first time,this study provided important evidence at the molecular level that the DNA fragment derived from the paternal parent occurred in GGC.Besides,this DNA fragment provided a suitable molecular marker for GGC to be distinguished from other GC species.(2)A total of 32 Hox genes and two extra DNA fragments(K522)that did not belong to Hox gene clusters were amplified from KOC,GGC,and GC by using nine pairs of primers(HoxA4a,HoxA9 a,HoxA11b,HoxB1 b,HoxC4a,HoxD10 a,HoxC6b,HoxC6b-G,and HoxC6b-K).Sequencing results showed that GGC had a recombinant gene(HoxC6b-GGC-2)thathad both parental genetic structures and preference to KOC.Besides,GGC also had HoxC6b-GGC which was the same to GC,and HoxC6b-GGC-1 which was the same to KOC.In addition,in the amplification results of GGC and KOC,the same DNA fragments of K522 that did not belong to Hox gene cluster had been amplified by the primer of HoxC6 b,respectively,and it was not amplified in GC.Sequencing results showed that the same fragments of K522 amplified in GGC and KOC had the same sequence.HoxC6b-GGC-2 was the first recombinant gene which was reported in GGC.This discovery provided further evidence for remains of paternal DNA and “hybrid effect” in gynogenetic progenies.(3)The sequences of mitochondrial DNA and 5S ribosomal DNA of KOC,GGC and GC had been amplified and sequenced to identify their homology.The results showed that GGC had high similarity with GC in structure of mitochondrial DNA and 5S ribosomal DNA.(4)The liver and spleen transcriptomes of GGC and GC were analyzed to reveal their different genes by using RNA-seq.The results showed that a total of 3145 unigenes were differentially expressed between GC and GGC,including 1666 upregulated and 1479 downregulated unigenes in GGC.GO enrichment and KEGG signal pathway enrichment were used to analyze these unigenes.The possible disease resistance mechanism of GGC was inferred according to these results.(5)In order to explore the barrier function between GGC and GC,the expression levels of nine tight junction protein related genes(occludin,claudin15 a,JAM,claudin2,claudin7 a,claudin4,ZO-1,cingulin and sympk)in GGC and GC were validated by quantitative polymerase chain reaction.The verification results showed that the expression levels of nine tight junction protein related genes in GGC were higher than GC,which consistent with the transcript analysis results.It indicated that GGC had the stronger barrier function than GC,which made GGC had higher anti-disease than GC.