Study On The Self-backcross Test And Cytology Of Hybrid F1 Gametes Between Misgurnus Anguillicaudatus And Paramis Gurnus Dabryanus

Dojo loach（Misgurnus anguillicaudatus）（assigned as DD,2N=50）and large scale loach（Paramisgurnus dabryanus）（assigned as PP,2N=48）belong to Cobitidae and Cobiinae.The artificial hybrid（assigned as DP and PD,2n=49）of these two loach species has been proved to perform degenerated gonadal development.The purpose of this study was to explore the fertility of F1 generation of hybrid between M.anguillicaudatus and P.dableurus by self-crossing and backcrossing.Ten mating combinations（♀×♂;DD×DD,PP×PP,PD×DD,PD×PP,DP×DD,DP×PP,DD×DP,PP×DP,PP×DP,DD×DP,PP×DP,DD×PD,PP×PD）were designed and the F1gametes of the hybrid between M.anguillicaudatus and P.dableurus were analyzed from the cytogenetics.The main research contents are as follows:1 Artificial fertilization of Self-Backcross test between M.anguillicaudatus and P.dableurus and comparison of growth difference of the progeniesIn this study,we found that there was different fertilization,hatching,deformity,and survival rate among these 10 crosses.We found that the fertilized eggs of the self-crossing and hybridization of M.anguillicaudatus and P.dableurus could develop normally.However,there were no normal fertilization eggs in the combinations using DP and PD as the male parent.The results showed that the fertility rate was PP×PP>DD×DD>DP×PP>PD×PP>DP×DD>PD×DD.Among them,the fertilization rate of the self-crossing combination DD×DD（70.92±2.22%）and PP×PP（83.06±1.97%）was significantly higher than that of the backcrossing groups（P<0.05）.In the backcross combinations,the fertilization rate of the backcross combinations using P.dableurus as a male parent was significantly higher than that of the backcross combinations using M.anguillicaudatus as a male parent.The date of relative hatchability was PP×PP>DD×DD>DP×PP>PD×DD>PD×PP>DP×DD,which didn’t show significant differences,and PP×PP（91.17±0.39%）showed the highest relative hatching rate.The relative malformation rate was statistically DP×DD>PD×PP>PD×DD>DP×PP>PP×PP>DD×DD,in which the two self-crossing combinations were significantly lower than that of the four backcrossing combinations.The result of the relative 7-day survival rate was PP×PP>DD×DD>DP×PP>PD×DD>PD×PP>DP×DD.Here,two self-crossing combinations were significantly higher than four backcrossing combinations.At the end of 1^stmonth,the purebreds PP×PP showed a significantly longer total length compared to other genotypes.The shortest total length was observed in genotype PD×DD but was at the same level with DD×DD,PD×PP,DP×PP,and DP×DD genotype（P>0.05）.At the end of the 4^thmonth,the growth of each genotype was different compared with the short-term growth trial.The PD×PP group with a total length of 84.6 mm and a weight of 7.24 g showed the best growth performance,while the DD×DD group with a total length of 70.86 mm and a weight of 4.85g showed the poorest growth performance.The PD×PP,PP×PP and DP×PP groups,with P.dabryanus as male parent,showed significantly better growth performance than DP×D,PD×D,and DD×groups,with M.anguillicaudatus as female parent,and the same trend was observed in body weight.2 Embryos development of self-crossing and backcrossing between M.anguillicaudatus and P.dableurus.Zygotes of six combinations PP×PD,DD×PD,PD×PP,PD×DD,PP×PP,and DD×DD were observed.Which showed no fertilization in PP×PD and DD×PD combinations with obvious characteristics of unzygotes.In contrast,Normal embryonic development of PD×PP、PD×DD、PP×PP、DD×DD could be observed in the experiment.Generally exhibiting common stage characters:Blastoderm stage;2-cell stage;4-cell stage;8-cell stage;16-cell stage;32-cell stage;64-cell stage;Morula stage;Early bastula stage;Late blastula stage;Early gastrula stage;Mid gastrula stage;Late gastrula stage;Neurula stage;Blastopore closing stage;Myomero formation stage;Eyesac formation stage;Tail bud stage;Rudiment of tail stage;Eye lens formation stage;Muscular contraction stage;Beating heart stage;Hatched larva;Larva When the water temperature was 25±0.5℃,the hatching time of PP×PP was 35 h 40 min;PD×PP,36 h 16 min;PD×DD,34h 46 min.The backcrossing combinations PD×PP and PD×DD were slightly longer than that of self-crossing combinations DD×PP and PP×DD.Overall,there was no significant difference in complete embryo development time between the four combinations.3 Karyotype analysis of backcrossing progeny between M.anguillicaudatus and P.dableurus.The karyotype analysis of backcrossing progeny of M.anguillicaudatus and P.dableurus was carried out by the cell culture method.It is found that the number of chromosomes in backcrossing progeny cells was 49 when both PD and DP were the female parent and DD was the male parent.The number of chromosomes in the backcross progeny was 48 when PP was the male parent.The karyotype formula is as follows:DP×DD（2N=49,10 m+6sm+33t,NF=65）;PD×DD（2N=49,11m+4sm+34t,NF=62）;DP×PP（2N=48,11m+6sm+31t,NF=65）;PD×PP（2N=48,12 m+4sm+32t,NF=64）.Through comparative analysis,it can be inferred that the reciprocal hybrid loach DP and PD produced fertile eggs containing 24 chromosomes with different chromosome composition（5m+4sm+15t and 6m+2sm+16t respectively）or Robertson translocation may have occurred during the process of gamete formation in meiosis of hybrid loach.4 Analysis of sperm motility,morphology,and structure in F1 generation of hybridization between M.anguillicaudatus and P.dableurus.The loach M.anguillicaudatus（DD）（Cobitidae;Cobitinae;Misgurnus）,and large scale loach P.dabryanus（PP）（Cobitidae;Cobitinae;Paramisgurnus）,hybrid F1 generation（DP）,and（PD）four loach sperm morphology and vitality are analyzed through computer assisted sperm analysis system（CASA）,optical microscope,scanning electron microscopy（SEM）,transmission electron microscopy（TEM）and flow cytometry technology in order to explore the reason of male sterility in diploid loach and large scale in vice loach hybrid F1 generation,preliminarily.The results showed that the sperm motility rates of DD and PP were 76.50±0.70%and75.17±8.60%respectively at 30s of activation,which were significantly higher than DP（3.65±1.75%）and PD（2.68±0.63%）（P<0.01）,and the related motion parameters of hybrid loach were also significantly lower than DD and PP（P<0.01）.Ploidy identification was carried out on the sperms of four species of lodges in the sperms of DD and PP by flow cytometers.It was found that the sperms of DD and PP were mostly 1N cell,while there were a large number of 2N and 4N and a small number of 8N cells in the sperms of DP and PD except for the normal haploid sperm.The spermatozoa in DD and PP spermatozoa were dense,intact and uniform in length（33.71±2.12）and（34.28±1.83）m,respectively,by SEM and TEM.DP and PD in the testis appeared a large number of abnormal sperm,such as multitailed sperm,binuclear sperm,and big-head sperm.Head size showed obvious polymorphism,according to the head size measurements to estimate its ploidy,including normal sperm haploid sperm,at the same time have a diploid and tetraploid sperm,or even higher haploid sperm.The abnormal sperm of these hybrid loaches provided important evidence for their low sperm motility and male sterility.