Wei Wen-Hui, Zhang Jing, Zhang Yi-Bing, Zhou Li, Gui Jian-Fang
State Key Laboratory of Freshwater Ecology and Biotechnology, Wuhan Center for Developmental Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430-072, China.
Cytometry A. 2003 Nov;56(1):46-52. doi: 10.1002/cyto.a.10077.
Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones.
The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4 degrees C. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 microg/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems.
Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5.38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis.
Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus).
多年来，通过核型和细胞学分析，在雌核发育的银鲫（Carassius auratus gibelio Bloch）中已鉴定出一些三倍体和四倍体克隆。此外，在其自然和人工种群中发现了5%-20%的雄性个体以及核型多样性。然而，DNA含量及其与倍性水平和染色体数目的关系尚未确定，不同克隆中精子发生过程中是否发生正常减数分裂也有待确定。
将采集的血细胞或精子与鸡或银鲫个体的血细胞混合，于4℃用70%预冷乙醇固定过夜。将混合细胞沉淀用1×磷酸盐缓冲盐水洗涤2-3次，然后重悬于含有0.5%胃蛋白酶和0.1 M盐酸的溶液中。用含有4 kU/ml核糖核酸酶的碘化丙啶溶液（40 μg/mL）对DNA进行染色。用Phoenix Flow Systems进行DNA含量测定。
三倍体克隆A、E、F和P的DNA含量几乎相等，但三倍体克隆D的DNA含量高于其他四个三倍体克隆。克隆M（7.01±0.15 pg/核）的DNA含量几乎等于克隆D（5.38±0.06 pg/核）的DNA含量加上鲤鱼精子的DNA含量（1.64±0.02 pg/核）。克隆A、P和D精子的DNA含量是其血细胞的一半，这表明精子发生过程中发生了正常减数分裂。
流式细胞术是分析多倍体银鲫不同雌核发育克隆间遗传异质性和倍性水平的有力方法。通过本研究，回答了四个问题。（a）揭示了银鲫中五个三倍体克隆和一个多倍体四倍体克隆之间的DNA含量相关性，其含量不仅随倍性水平增加，也随染色体数目增加。（b）克隆D的六条额外染色体的平均DNA含量为0.052 pg，高于克隆A、E、F和P中每条染色体的含量（约0.032 pg/染色体）。这意味着这六条额外染色体是较大的染色体。（c）银鲫精子发生过程中发生了正常减数分裂，因为克隆A、P和D精子的DNA含量几乎是其血细胞的一半。（d）多倍体四倍体克隆M（7.01±0.15 pg/核）包含克隆D（5.38±0.06 pg/核）的完整基因组和鲤鱼精子的基因组（1.64±0.02 pg/核）。
