FISH Analysis On Germ Cells In Hybrid Strain Of Red Crucian Carp × Common Carp

Distant hybridization refered to the hybridization between different species or between genera or even above families,and was one of the important ways to improve and cultivate new species.In the intergeneric hybridization of red crucian carp(Carassius auratus red var.,♀,abbreviated as RCC)×common carp(Cyprinus carpio L.,♂,abbreviated as CC),they were artificially hybridized to produce viable diploid hybrid F1 offspring,in which some males and females were fertile and mated each other to produce F2,F2 self-crossed to produce a hybrid F3 generation.In the hybrid F3 individuals,bisexual fertile allotetraploid fish(4n=200)had been detected out,which has been continuously bred for 27 generations and formed a new strain(F3-F29),namely Allotetraploid hybrid fish of red crucian carp ×common carp,abbreviated as 4nAT.Hybridization of 4nAT(4n=200,(♂)× improved red crucian carp(2n=100,♀)can obtain a large-scale sterile improved triploid crucian carp,called Xiangyun crucian carp 2(3n=150,abbreviated as TCC).In this study,the germ gland cytological structure and FISH analysis of germ cells of F1,4nAT and TCC were systematically studied on the premise of collecting a large number of experimental fish at different developmental stages,and the hybrid parent RCC and CC were used as the control.The meiotic cytological process of the hybrid strain of red crucian carp × common carp was comprehensively discussed.The main contents of germ cell FISH analysis are as follows:1.Chromosome FISH analysis was performed on the hybrid strain of red crucian carp × common carp and their parents using 340-bp 5S rDNA sequence(340-bp,GQ485556)of RCC as probe.This probe was a specific probe for crucian carp and only has obvious fluorescence signal on the crucian carp chromosome group.FISH study of germ cells in RCC and 4n AT demonstrated that the strong specific fluorescence signals appeared in pair at the prophase of MI.In the FISH study of F1 spermatogenic cells,the chromosome spread in the first meiotic prophase was composed of rod-like structure and filamentous structure,in which a single signal was showed on the undoubled chromosomes spread.After chromosome duplication,the prophase and metaphase of doubled primary spermatocytes showed paired double signals,presumably the chromosome pairing ourring between chromosome of crucian carp and crucian carp,commoncarp and common carp.Furthermore,the paired signals consisting of RCC chromosomes can be seen in TCC.2.Chromosome FISH analysis was performed on on the hybrid strain of red crucian carp × common carp and their parents using the unique centromeric repeats(263-bp,sequence number JQ086761)of RCC as probes.The probe could also clearly distinguish the chromosomes of crucian carp and common carp.In the FISH study of F1 in spermatogenic cells,the chromosme locations in undoubled cells appeared partially pairing,in which the chromosomes possessed uneven with rod-shaped areas and filamentous areas.In the FISH study of spermatogenic cells in 4nAT,50 bivalents of 100 bivalents had paired signals,with the other 50 bivalents had no signals,which presumably showed that 50 bivalents originated from chromosome pairing of red crucian carp and 50 bivalents originated from chromosome pairing of common carp.The results showed that FISH analysis on germ cells in hybrid strain of red crucian carp ×common carp,could duplicate chromosomes by the characteristics of chromosome pairing in centromere region,and the chromosome pairing of reproductive cells after replication occurred between red crucian carp and red crucian carp,and between common carp and common carp.In addition,it was found that the 263-bp centromere repeat sequence was not located in the middle area of the bivalents,indicating that there might be a special meiosis pairing structure in fish to be further analyzed.In addition,in the cell phase of nearly 150 chromosomes in TCC,about 80 chromosomes carrying single signal appeared in pairs,which proved that there was a high frequency of chromosome pairing between crucian carp and crucian carp in TCC.3.The pairing trend of F1 female oocytes was significantly better than that of male spermatocytes.In the early stage of meiosis I reduction,the oocytes without chromosome doubling showed relatively uniform filamentous structure.A strong signal and some weak signals could be seen in 5S rDNA sequence location.After doubling,the filamentous structure was more uniform,and the 5S rDNA sequence location signal was doubled.The location characteristics of 263-bp centromere repeat need to be further clarified.This study systematically discussed the meiosis process on germ cells in hybrid strain of red crucian carp × common carp,and the genetic pairing characteristics of allogeneic chromosomal groups in the process of hybrid fish breaking through reproductive difficulties,which will provide important guidance for the study of distant hybridization of fish,and have important significance in the theory and practice of fish genetic breeding.