Establishment And Main Biological Characterization Of Gynogenetic Grass Carp Population

The grass carp is one of an important fish cultured in china. However, farmed grass carp are readily susceptible to various kinds of diseases that cause high percentages of morbidity and mortality, leading to greatly reduced yields. Gynogenesis is considered to be an efficient way to rapidly establish pure line. Gynogenesis, in combination with selective breeding, has been suggested as a shortcut to produce clonal fish lines with desired characteristics, such as higher growth rates and disease resistance. The establishment of pure all-female groups of meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG) grass carp should be a valuable contribution to both fish genetics and grass carp breeding.Grass carp (Ctenopharyngodon idellus) eggs were first activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (?) X common carp (?) and then duplicated by temperature shock (cold shock or heat shock). Different shock initiation time resulted in two types of diploid gynogenetic grass carp:meiG and mitG. The fertilization rate,hatching rate and survival rate of both gynogenetic fish was highly improved after optimizing gynogenetic technique. Over a five year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. Now two groups of gynogenetic fish in large scale had been successfully established. The main biological characteristics of gynogenetic grass carp were studied, and their growth, development process of their gonad were seriously observed for several years. The major results are as follows:1. UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (?) X common carp (?) was first used for inducing gynogenesis in grass carp and showed high efficiency. The protocols for eggs duplication were optimized and high survival rate of both meitG and mitG were obtained. Over a five year period, both meiG and mitG group were successfully established in large scale.2. The observation of early embryo development confirmed that haploid embryos with haploid symptoms were inviable, while diploid gynogenetic grass carp embryos possess of natural embryo development stages and morphological characteristics as normal diploids. Incubating time was influenced by hatching temperature. On the other hand, water dissolved oxygen affecting the hatching rate and survival rate of gynogenetic fish directly.3. Gynogenetic grass carp had identical morphological characteristics as common grass carp. The size and morphological traits of red blood cells also similar in gynogenetic fish and common grass carp, which indicating that the gynogenetic fish were diploids same as common grass carp. It was confirmed by further chromosome examination of embryos and young gynogenetic fish.4. Microsatellite DNA analysis indicated that the genome of gynogenetic fish were not contaminated by paternal genome, the genetic purity of mitG grass carp was higher than that of meiG grass carp, and both of which were significantly increased over that of common grass carp after one generation of gynogenesis. A special SCAR marker was successfully transferred from RAPD marker, which could be used for identify gynogenetic grass carp from common carp.5. The growth rate polarized distinctively in gynogenetic grass carp group after 5 years track. Gonads observation showed that both meiG and mitG grass carp groups were 100%female and 88%of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The all-female gynogenesis larvae of grass carp were fed with food containing 17α-methyltes tosterone for 4 months. The histological observation results indicating that the inducing of sex-revesal may success.6. A Foxl2 cDNA was cloned from meiG ovary by RT-PCR and subsequent RACE. Alignment of known Foxl2 sequences from vertebrates confirmed the conservation of the Foxl2. RT-PCR revealed that Foxl2 is expressed in the grass carp brain (B), pituitary (P), gill, and gonads (G), reflecting the involvement of Foxl2 in B-P-G axis. Large-scale scan in gynogenetic fish and common grass carp showed that the Foxl2 only express in ovary while not express in testis, which revealed en evident sexual dimorphic expression pattern in the gonads. This traits could be used for identify the sex of grass carp from molecular level.7. The complete mitochondrial genomes of gynogenetic grass carp was successfully sequenced and analyzed. The results indicated that gynogenetic grass carp obeys the maternal inheritance rule, and the inheritance structure of their mitochondrial DNA is identical to that of common grass carp.