Studies On The Chromosome Of The Silkworm And Chromosome Assignment Of A Novel ISSR Polymorphic Marker By Fluorescence In Situ Hybridization

Classification and morphology reseaches of chromosome were once the key reseaches of genetcis.Karyotype analysis had became one of the most important research tools in genetic polymorphism,which can distinguish chromosomes from each other by contrasting configuration parameters such as relative lengths, centromere position and chromosome bands. However, Lepidopteran species had a relatively high number of small holocentric chromosomes (Bombyx mori: 2n = 56) in metaphase. Chromosome identification had long been hampered in this group owing to the high number and the absence of suitable markers like centromere position and chromosome bands.Therefore, in the early days, karyotype analysis and gene mapping were primarily done by making use of chromosomal relative lengths and particular strains, such as the sex-limited or transnational strains. And the research materials were often meiotic pachytene chromosomes of sex gland and mitotic prometaphase of early embryos and diapause eggs' somatic cells. Now we have found that all materials and methods early used have some limitations.Fluorescence in situ hybridization links the cytogenetics and molecular genetics.It can determine the exact loci of genes on chromosomes or chromatin.When it was introduced into the researches of silkworm, it had resolved many difficult problems such as chromosomal structure, gene mapping, karyotyping,etc.Therefore, we should develop new materials and use new methodology to study the genetcis of silkworm.Based on former researches obtained from other scientists, this research aimed to obtain the meiotic pachytene modle karyotype of silkworm by new metrical software; study the characters of mitosis in the cultured cells of the silkworm.And on the basis of former karyotype, we used inter simple repeated sequences (ISSR) as probes for fluorescence in situ hybridization (FISH) on chromosomes prepared from B. mori females. The result were as follows: 1) Modle karyotype analysis of meiotic pachytene of silkwormFive varieties of silkworm were investigated.From each of the silkworm, 5 meiotic cells with good characters for karyotyping were selected to measure the length of chromosomes. Then we got the average value of absolute length of the pachytene chromosomes.On the basis of these results, we established the karyotype model of the pachytene chromosomes.It showed that in the karyotype of the pachytene chromosomes, the length of each variety with the same sequence number had no evident distinction. Not only the total absolute length was near to each other, but also a number of SD values were bigger than 1. Relative length was the ratio between one chromosme length and total length of 28chromosomes. The karyotype established by relative length could avoid some difficulties resulting from absolute length. In this karyotype, there were no distinct differences between each variety with the same sequence number.The biggest SD value was 0.144, while the smallest was 0. 032. This indicated that karyotype established by relative length was better than by absolute length.The longest chromosomes were 2.5 times longer than the shortest ones, not only in relative length but also in absolute length.For the karyotype analysis,we could conclude that chromosomes in the later pachytene were uniform among different varieties of silkworm, and this karyotype was more uniform for gene mapping.In this work, we used Image J to measure absolute length.This method was better than traditional method, since it could reduce steps, decrease error and improve efficiency.2) Mistosis research of cultured cells of the silkwormThe research firstly recorded miototic process in the cultured cells. When the primary cells were cultured for a certain time, they could proceed normal miototic process like insect and mammalian continuous cell lines.The cells carrying through mitotic prophase shrank to be round, became more stereo,had finished replication of DNA ,and were bigger than other nonmitotic cells.Chromosomes got together when mitotic prometaphase was taking place.In the metaprophase.all the chromosomes assembled and arrayed on the plate of cell equator. When the anaphase came, the chromosomes lined on the cell plate separated into two clusters, and the morphology of the cells presented dumbbell-like structure. In the later telophase, chromosomes became invisible,and in the interphase mother cells divided into two daughter cells.The split ratio ranged from 0.02±0.01% to 0.35±0.10% when the primary cells were cultured for 6 and 10 months reapectively.The division split ratio of the continuous cell line was 16.5%.The nucleus of cells contained numerous chromosomes.The chromosomes were counted under a microscope randomly with 155 samples. The frequencies of chromosome numbers appeared to follow a normal distribution (Fig. 4). The statistical analysis of the karyotype of the cell line indicated that this cell had a great variation in chromosome number. A giant cell was found to contain 339 chromosomes. It was difficult to get a modle karyotype.The mitotic metaphase chromosome were mostly short-pole and granule-like.The longest metaphase and prometaphase chromosome was respectively 4.505μm and 7.965μm, and the shortest was respectively 1.078μm and 1.068μm. The length of the longest prometaphase chromosome was near to the middling pachytene, and about two times longer than that from early embryo. The diversities of the prometaphase chromosome were more distinct from each otherFor these reasons, the prometaphase from cultured cells was good material for gene mapping.In the research, we also found the abnormality of the cell plate and chromosome...