| Grass carp(Cetpharyngodon idellus)is one of the most unique freshwater aquaculture species in China,which plays an important role in the breeding of fish.As the grass carp breeding cycle is long,broodstock individual and other reasons,to the application of traditional breeding methods to produce fine varieties of certain difficulties,there is no state for examination and approval of the grass carp at present.Gynogenetic development is an effective way to establish pure lines quickly.It is possible to evaluate the genetic diversity of gynogenetic progeny by microsatellite markers,and it may be feasible to carry out seed breeding on the basis of good performance of the Yangtze River water grass carp.This laboratory established 6 age ENU mutagenesis grass carp group,and four pairs of excellent parents were selected to establish family,we used the mstn1 and mstn2 gene polymorphisms to study the growth traits of four families in order to select candidate molecular markers for the improvement of population growth of ENU mutagenesis grass carp,and to further identify the molecular marker-assisted breeding program of ENU mutagenesis grass carp population.Specific research is as follows:(1)After activated by the UV inactivated sperms of blunt snout bream,we induced the gynogenetic offspring of the excellent Yangtze River grass carp by cold shock inhibiting second polar body method.There were gynogenetic grass carp and grass bream hybrids in the offspring.The size of gynogenetic grass carp was consistent of the grass carp,while the size of grass bream hybrids was between grass carp and blunt snout bream.Partec flow cytometry showed that the relative DNA of grass carp and gynogenetic grass carp was 23.01 and 22.72,of which the two groups were close;the relative DNA of high size offspring was 25.38 between the grass carp and blunt snout bream,which should be the grass bream hybrids.17 microsatellite markers were selected to detect the genetic diversity of the grass carp group,the gynogenetic grass carp group and the grass bream hybrids.The results showed that 59 alleles were detected,of which 43.18 were effective alleles.The mean value of number on alleles(Na)of the grass carp,the grass bream hybrids and the gynogenetic grass carp group was 3.57,2.86 and 2.79;the mean value of the effective alleles was 2.93,2.37 and 1.96;the mean value of expected heterozygosity(He)was 0.6502,0.5573 and 0.3775;the average value of polymorphism information content(PIC)was 0.5738,0.4649 and 0.3791,respectively.Compared with the grass carp control,the genetic diversity of the gynogenetic grass carp decreased significantly,indicating that meiotic gynogenetic method could get high homozygous of gynogenetic grass individuals.We modeled the Microsatellite DNA fingerprintings of the different grass carp group and screened out nine specific microsatellite markers.This study will provide the basic molecular genetics information of the excellent grass carp.(2)In order to obtain genetic parameter of gynogenetic ENU mutagenesis grass carp(Cetpharyngodon idellus)group,the research showed that the relative DNA contents of the ENU mutagenesis grass carp and the gynogenetic ENU mutagenesis grass carp were 24.02 and 23.80,which were close by using Partec CyFlow ploidy analyzer and they were diploid.28 microsatellite markers were selected to detect the genetic diversity of the ENU mutagenesis grass carp group(Q group)and the gynogenetic ENU mutagenesis grass carp(E group).The results showed that the average alleles of E group and Q group were 3.7143,5.1786,the average effective alleles were 2.1857,4.0028,and the values of average expected homozygosity were 0.5122,0.2814,and the values of average expected heterozygosity were 0.4878,0.7186,and the values of average polymorphism information content(PIC)were 0.4282,0.6606.In terms of the homozygous rate analysis of individual in the microsatellite loci,each individual's degree of purity was less than 1.00 in the E group,indicating that there was not completely homozygous individuals.In terms of each microsatellite loci in groups of homozygous rate analysis,in addition to the microsatellite loci 5476,and HLJC81 HLJC118,other locis of homozygosity were improved significantly at different rates.To sum up,though meiotic gynogenesis method the homozygosity of each microsatellite site in the ENU mutagenesis grass carp group was improved at a different rate and the genetic diversity was decreased significantly,this method can only obtain individuals of gynogenetic ENU mutagenesis grass carp with high homozygosity and provide important genetic data for the excellent selection of ENU mutagenesis grass carp.(3)Four ENU mutagenesis grass carp families were grown and compared,and after 175 days of breeding,from the point of view of weight gain,family 1 and family 4 have obvious advantages over the other two families.From the weight gain rate,family 4 and family 2 have obvious advantages compared with the other two families.Family 4 has the most obvious advantages,family 3 was weaker than the other three families.The partial correlation analysis showed that the relationship between the morphological traits and the body weight was obtained: Family 1 included full length,body length,tail handle length,body width,correlation coefficient were 0.357,0.619,0.608,0.619;Family 2 had full length,body length,head length,thickness,the correlation coefficient were 0.348,0.360,0.687,0.360;Family 3 included full length,body length,shank length,shank height,correlation coefficient were 0.529,0.449,0.351,0.384;Family 4 had full length,body length,body thickness,the correlation coefficient were 0.629,0.543,0.590.The above statistical methods can be used to summarize the morphological differences between family 4 and family 3.The mstn1 and mstn2 genes were screened by SNP loci in families 3 and 4.The SNP loci were screened in MSTN1 and MSTN2 loci by using two-way sequencing.For MSTN1,465 nt C / G and 467 nt G / A in family 3,465 nt C / G in family 4 are found missense mutations;For MSTN2,912 nt C / T in family 3 and family 4 is found synonymous mutation,1027 nt G / A in family 3,366 nt A / G and in family 4 are found missense mutations,and SNP sites were found in both noncoding regions 1390 nt A / T and 1401 nt G / A in both families.These results indicated that the SNPs of MSTN1 and MSTN2 genes are closely related to the growth traits of ENU mutagenesis grass carp. |
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