| Haliotis discus hannai(hereinafter referred to as D)was an important breed of abalone in China,H.fulgens(hereinafter referred to as F)was a kind of abalone imported from California,USA.In 2010,the interspecific hybridization technique of H.discus hannai and H.fulgens was breakthrough,and the hybrid F1 of DF and FD showed heterosis in the survival rate,growth speed and high temperature tolerance,which had good prospects for application.However,the cytogenetic method of hybrid identification of H.discus hannai and H.fulgens was still unstable,and the genetic characteristics of repetitive sequences have not yet been carried out.Therefore,this study aims to optimize the GISH(genomic in situ hybridization)to make it more suitable to study the hybrid abalone,on this basis,the chromosome structure of hybrid F1 between H.discus hannai and H.fulgens was analyzed.In addition,cloning,sequencing,sequence alignment and FISH(Fluorescence in situ hybridization)analysis of 45 S rDNA fragments(including ITS1-5.8S rDNAITS2)of H.discus hannai,H.fulgens and their hybrids were carried out.The main results were as follows:1.The optimization experiment showed that when GISH was used in the study of hybrid abalone,the suitable range of the main components of the hybridization buffer was as follows: the length of the probe was 250~500bp,the amount of the probe was 62.5~125ng/10μL,the dosage of the blocking salmon DNA was about 10 times the amount of the probe,and the concentration of deionized formamide in the hybridization buffer was about 30%,the concentration of dextran sulfate(DS)or polyethylene glycol(PEG)6000 was about 12.5%~17.5% or about 7.5%.2.Karyotype analysis showed that the hybrid F1 of DF and FD all contained 36 chromosomes,and their karyotypes were 2n=22m+12sm+2st,NF=72.GISH analysis showed that the hybrid F1 cell of DF contained one set of chromosomes complement from male parent and female parent respectively.3.The 45 S rDNA fragment(ITS1-5.8S rDNA-ITS2)sequence alignment analysis showed that there were 26 SNP markers and 12 In Del markers in the ITS1 and ITS2 regions,of which 16 SNP and 10 InDel markers could distinguish between H.discus hannai and H.fulgens.Moreover,the sequences of DF and FD hybrid F1 were all similar to their female parent.Specific primers designed to amplify the specific ITS of H.discus hannai or H.fulgens,PCR analysis showed that both DF and FD hybrid F1 contained the specific ITS of H.discus hannai and H.fulgens.4.The FISH location results of 45 S rDNA fragment(ITS1-5.8S rDNA-ITS2)showed that there were 3 pairs of signals located at the end of the parental chromosomes.but the chromosomes with positive signals were slightly different: 3 pairs of signals were located on the m chromosome of H.fulgens,and H.discus hannai contained 2 pairs of signals on metacentric chromosome and 1 pairs of signals on submetacentric chromosome.Both hybrid F1 of DF and FD have 6 45 S rDNA gene clusters,which were distributed on 5 metacentric chromosomes and 1 submetacentric chromosome.In conclusion,this study systematically optimized the main components concentration(dosage)and other parameters of the hybridization buffer in the GISH which used to study hybrid abalone,those results improved the controllability of GISH.However,the difficulty of identifying parental chromosomes in DF and FD were still far higher than that in the hybrid F1 of H.diversicolor diversicolor and H.discus hannai.This result indicated that the homology of the long distant species H.discus hannai and H.fulgens was much higher than the neighboring species H.discus hannai and H.diversicolor diversicolor.There was interspecific differentiation in the 45 S rDNA ITS sequence,and the hybrids contained 45 S rDNA from parents,but the results suggested that the hybrid 45 S rDNA may have the characteristics of maternal bias.It can be seen that the typing of SNP or In Del markers of 4S rDNA ITS can be used to identify the genetic composition.In addition,the maternal bias of 45 S rDNA sequences of hybrid F1 was worth further studying. |
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