Efficient Production of Doubled Haploids as Isogenic Line Founders via Double Heat Shock Treatment in Willow Gudgeon (Gnathopogon caerulescens)

Figure 4

Nine patterns of microtubule organisation and nuclear alteration after HST2, estimated from varying degrees of centriole destruction via HST during the first cleavage, and ploidy of hatched larvae. (a) Normal development in intact control eggs (IC). The two centrosomes are not damaged, and occurred normal chromosome separation, forming normally developed diploids (normal 2n). (b)–(i) Gynogenetic development. (b) Untreated gynogenetic control (GC), eggs produced via insemination of UV-irradiated sperm. Sperm-derived genetic material (dense chromatin body, dcb, and red rectangles) cannot be expanded into chromosomes. Only chromosomes from the egg nucleus (grey rectangles) show normal segregation and form haploids (G n) because the two centrosomes are intact. (c) One of four centrioles in two centrosomes is depolymerised by HST2. Normal chromosome separation is achieved in the first cell cycle; however, in the second cell cycle, one blastomere yielding one completely duplicated centrosome and a monopolar spindle and another blastomere yielding two complete centrosomes and a normal bipolar spindle are formed, producing an n/2n mosaic (Mito-G n/2n mosaic). (d) One centrosome is completely depolymerised, and the other remains intact; in the first cell cycle, a monopolar spindle is formed, chromosomes do not separate, and the process is completed without cell division, yielding a doubled haploid nucleus (DH 2n). (e) The daughter centrioles of both centrosomes are depolymerised, and each mitotic pole is formed by one centriole. In the first cell cycle, chromosome division usually proceeds only in the egg nucleus-derived chromosomes. In the second cell cycle, both spindle poles form the unipolar spindle and two doubled haploid nuclei (DH 2n). (f) Only one centriole remains and forms a monopolar spindle in the first and second cell cycles, which gives rise to a quadruple haploid of the homozygote (QH 4n). (g) In the untreated gynogenetic control (GC) eggs, when spontaneous retention of the second polar body (SR-2PB) occurs, the egg nucleus becomes diploid, and the dcb derived from UV-sperm cannot be expanded into chromosomes. Additionally, chromosomes from the diploid egg nucleus achieve normal segregation and form meiotic gynogenesis diploids (Meio-G 2n) because the two centrosomes are intact. (h) After SR-2PB, one centrosome is completely depolymerised by HST2, and the other remains intact; in the first cell cycle, a monopolar spindle is formed, chromosomes do not separate, and the process is completed without cell division. Therefore, the heterozygote diploid female pronucleus is doubled, yielding a meiotic tetraploid nucleus (Meio-G 4n). (i) After SR-2PB, the daughter centrioles of both centrosomes are depolymerised, and each mitotic pole is formed by one centriole. In the first cell cycle, chromosome division usually proceeds with the heterozygous diploid egg nucleus-derived chromosomes. In the second cell cycle, both spindle poles form a unipolar spindle and two heterozygous meiotic tetraploid nuclei (Meio-G 4n). Activation, egg fertilisation; Ap, approach of both pronucleus; Z, zygote stage; P, prophase; PM, prometaphase; M, metaphase; A, anaphase; T, telophase.