| Carassius auratus is an important freshwater aquaculture species in China. Its variants are rich, and their reproduction mode is complex. In production, the main way is to use other species' sperms to stimulate the development of the eggs, and produce stocks with enhanced traits by gynogenesis. However, after generations germplasm resources recession would occur due to the low rate of variation in gynogenesis. In addition, the low rate of variation in heterologous sperm stimulated gynogenesis also may lead to the limitation of the survival time of those varieties. In order to improve the genetic diversity of parent populations and obtain more excellent traits, here we used microsatellite markers to developed Multiplex PCR system, accurately and efficiently exploring the genetic background of offsprings from different commercial strains, and the pattern of transmission of genetic material in filial generation and its effect on offsprings, so as to open up a new way for breeding of good varieties of crucian carp.In this study, we systematically detected the existed microsatellite markers, and redesigned primers according to the need of product range. And through multiplex PCR amplification tests, we established 2 sets of six markers, 1 set of 13 microsatellite loci, and 1 set of 15 microsatellite loci, and used these Multiplex PCR reaction systems to analyze the genetic structure and genetic relatives of 15 commercial strains (from different seed fields of Jiangsu, Henan, Hubei and Jiangxi commercial strains, A, D, E, nominal No. 3; B,F,G,H,I,J,K,L,M,nominal gibel carp; N,P,R nominal 2n Peng Zeji),and one wild population of Fangzheng Silver crucian carp (C, from Heilongjiang).The results showed that wild population C (average allele number, genotypes and observed heterozygosity were 7.0, 5.4, 0.68, respectively),Hong Zeyi F (6.5, 5.1, 0.80) and Dafeng crucian carp G (5.3, 3.6, 0.90) had the higher genetic diversity, while other population genetic diversity was low (average 3.9, 2.3, 0.31).The cluster analysis showed that wild populations of C was in the outgroup,maintaining a larger genetic distance with other commercial lines; N, P, R of Pengze crucian carp had a close genetic distance, No. 3 (A, E and D) did not cluster into one group,and the genetic differentiations of A, D, and B; E and H, and K, L and I, were very low.The boundary of so-called "Zhongke No. 3","Pengze crucian carp" and "crucian carp" is unclear, and the genetic relationship between nominal field sources is rather complex.For the family analysis, the segregation patterns of 13 loci in Multiplex PCR system were analyzed in the progeny of the parental families. The results showed that male DNA insertion were detected in 13 loci, and in all 13 families, the average insertion rate was 22.2%, significantly higher than that of heterospecific sperm stimulated families. Besides,we found that the genetic type of the parental self does not conform to Mendel segregation ratio, but showing the female parent genotypes, male and female genotype combination,and the super parent genotype. The genotypes of 77.8% individuals were same with female parent, showing the gynogenetic characteristics. There were three types of Male DNA insertion: 1) female homozygotes, accounting for 16.5%; 2) offspring genotypes showed combination of male and female, accounting for 2.9%; and 3) 2.8% offspring had no parental genotypes. The proportion of male DNA insertions in different loci was 0.59(HLJY16) to 0.01 (YJ30), and 0.361 to 0.09 in different families, both were significant(p<0.05), while the proportion of gynogenesis in all families was not significant (p>0.05).Notably, there is a considerable proportion of male offsprings in the next three generations.Compared with the female offsprings, the male parent DNA insertion rate was lower in the male offspring genotype, whether it was affected by the less sample number or other reasons, which still needs further explorations. |
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