Primary Research Of Allopolyploid Loach Induction And Its Fertility

Dojo loach(Misgurnus anguillicaudatus) and large scale loach(Paramisgurnus dabryanus) were belonging to Cobitidac, Cobitinae, Misgurnus and Paramisgurnus, respectively. They are two economic fishes in China. Loach was regarded as an excellent animal model to explain fish polyploidy origin and evolutionary because of its complex ploidy composition and abnormal gametic formation. This research conducted a series of allopolyploid loach induction using diploid Dojo loach(DD), tetraploid Dojo loach(TT) and large scale loach(PP). The growth performance, goand development and gamtic fertility of allopolyploid loaches were the main research contents in this study. And now will report the main results as follows: 1. Allopolyploid loach induction and fertilization cytology observationSix kinds of allopolyploid loaches(DD×PP-0, DD×DD-0, DD×TT-0, TT×PP-0, TT×DD-0 and TT×TT-0. The female parent in the former and the male parent last. 0 represent inhibited the second polar body released to obtaining triploid type progenies) were inducted using cold shock in this study. The cold shock condition was: 5 min after fertilization, 2-3 ℃cold water was used to induct allopolyploid progenies for 30 min. Testing statistical analysis of ploidy composition of different allopolyploidy loaches was conducted at different development periods. At one-month stage, induction success ratio of six kinds of allotriploid type loaches were 87.50%(DD×PP-0), 53.85%(DD×DD-0), 90.91%(DD×TT-0), 84.96%(TT×PP-0), 76.92%(TT×DD-0), 12.5%(TT×TT-0), respectively. At 12-month stage, the ploidy compositions of these loaches were similar to 1-month stage, the success ratio decreased with increasing ploid level of progenies. While, fertilization cytology observation confirmed that cold shock could inhibit the zygote’s second meiosis, and the second polar body was reserved to obtain allotriploid type progeny. 2. Growth performance and gonad development of allopolyploid loachGrowth performance of different inducted allopolyploidy progenies was analyzed by statistical analysis. There was not a significant difference in growth performance in the early life history, but in later life. At 12 months old, the progenies of pentaploidy(TT×PP–0) grow fastest than others. Little difference was observed in various ploidy level progenies in the same induction groups, except for pentaploid progenies grow slower than the triploid progenies in TT×DD-0 group. At the same time, there was a significant difference in the gonad development and gonad tissue cells ploidy composition among different allopolyploid loaches. Ovary contained 2N and 3N secodary oocyte, and testis contained 2.5N secondary spermatocyte and 5N primary spermatocyte of DD×PP-0 allotriploid progenies. Ovary contained 2N secodary oocyte and 4N primary oocyte, and testis contained 3N secondary spermatocyte and 6N primary spermatocyte of DD×DD-0 triploid progenies of. Ovary contained 2N secodary oocyte and 4N primary oocyte, and testis contained 1N spermatid and 2N secondary spermatocyte of DD×TT-0 allotetraploid progenies. Ovary contained 4N secodary oocyte, and testis contained 3N spermatid and 6N secondary spermatocyte of TT×PP-0 allopentaploid progenies. Ovary contained 4N secodary oocyte, and testis contained 2.5N spermatid and 5N secondary spermatocyte of TT×DD-0 allopentaploid progenies. Ovary contained 6N secodary oocyte of TT×TT-0 hexploid progenies. The gametic ploidy level did not conform to the conventional law of meiosis. The abnormal gametic formation of allopolyploid loaches was mainly including univalent missing and homologous chromosome pairing. 3. Fertility study and gametic formation mechanism of allopolyploid loachAllotriploid(DD×PP-0) and allopentaplod(TT×PP-0) loaches were used in this research for learning the fertility and gametic formation of allopolyploid loach. The gametic ploidy composition of allotriploid and allopentaplod loaches’ ovary was simple, but unlike the somatic cells ploidy. There were few fertile mature eggs in these two loaches. While, the testis of allotriploid and allopentaploid loaches were delayed development with abundant secondary spermatocyte cells, they were sterile. Gametic ploidy composition of these two loaches’ testis was complex, unlike the somatic cell. Allotriploid loach female mainly produced haploid mature eggs. While, allopentaploid loach could produce a variety of different ploidy gametes, most of them were diploidy, and a small amount of them were triploidy and aunoploidy. Tt was also confirmed that the allopentaploid loach can produce diploid second polar body during the second meiosis by cold shock result. 4. Gynogenetic characteristic of three kinds of tetraploid loachGynogenesis was conducted in wild tetraploid loach(N-TT), selfing breeding tetraploid loach(A-TT) and artificial induction tetraploid loach(M-TT), which stimulated by Megalobrama amblycephala inactivated sperm and without any treatment for chromosome doubling. There were detected 86.66~88.80% diploid and 11.20~13.34% tetraploid progenies in all three kinds of tetraploid loach gynogenetic offsprings, and tetraploid progenies can survive to adult fish. This automatic chromosome doubling phenomenon happened after fertilization, may be occur during the second meiotic division or the first mitosis by microsatellite analysis of paternal parent and microsatellite genotyping analysis. DNA was replicated and did not separated. This was the first time to observed that the zygote automatic chromosome doubling after fertilization in fish.