The Karyotype Analysis And Location Studies Of SSR On The Chromosomes Of Silkworm(Bombyx Mori.)

Chromosomes are the carriers of genetic substances.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 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 translocational strains.And the research materials were often meiotic pachytene chromosomes of sex gland and mitotic prometaphase of early embryos and diapause eggs' somatic cells.With the development of modem biology techniques,molecular biology techniques are now being increasingly applied,in studies on chromosome.Fluorescence in situ hybridization(FISH)has become a bridge between cytogenetics and molecular genetics, which enable us to do researches more detailedly at molecular level.Based on previous studies,this dissertation is to carry out FISH to determine the location of Simple Sequence Repeats(SSR)on silkworm chromosomes.The results are summed up as follows:1.The model karyotype of silkwormPachytene chromosomes from silkworm variety Dazao oocytes were investigated.10 meiotic chromosomes with clear configuration and intact amount were selected.Karyotype analysis softwares Image J were used to measure the length and carry out karyotype analysis.Results showed that there was a relative big distinction of total absolute length among different cells,the average value of chromosome total length of 10 cells was 311.903μm and the longest was 388.869μm,the shortest was 267.884μm,which showed a margin of 120.985μm.In the same cell, the absolute length of each chromosome was also different,the ratio between the longest and the shortest was 3.881,the average ratio was 3.007;the absolute length with same sequence number in different cells also showed a obvious distinction.For example,in 10 cells,as far as the second chromosome was concerned,the shortest was 12.924μm,while the longest was19.928μm,showing a margin of 7.004μm.Consequently,absolute length was unfit to be a parameter in silkworm karyotype analysis.The relative length with same sequence number in 10 cells was relatively close, the highest SD value was 0.889,while the lowest was only 0.095.Compared with absolute length, relative length has smaller difference.In this study,the data of absolute length and relative length of chromosome was compared with previous data.On the absolute length of each chromosome,the average value showed greatest margin of 2.163um and smallest margin of 0.957um.The relative length has smaller difference which showed greatest margin of 0.494um and smallest margin of 0.003um.we also did variance analysis of relative length of each chromosome through comparing data with previous studies.The results showed that there was no significant difference on the same number chromosome data and relative length of chromosome was a stable parameter which represents a proportion of a chromosome in entire chromosome group.It can avoid the differences which occurred in different period.This well prove that relative length of pachytene chromosomes was a stable data and it was more appropriate to act as a parameter to establish pattern on the condition of with same material.Finally,the above parameters were integrated and we established a model karyotype of meiosis chromosomes of silkworm,Bombyx mori,which partly reflected own characters of each chromosome.Compared with traditional length-measurement method by brass wires,the Image J softwares have superiority in measurement,for they reduce steps and subjective error.Especially in karyotype analysis,we don't need cut and paste pictures,which undoubtedly improve efficiency and veracity.2.FISH studies of SSR on the chromosomes of silkwormSSR was labeled with DIG and used as a probe,FISH was carried out on different stage chromosome preparations from silkworm and chromosomes were counterstained by DAPI.In both male and female silkworms,yellow signals of SSR(2301)and SSR(2310)were found on interkinesis,pachytene,metaphase chromosomes.Compared with mammals,signal checking percent was lower.Hybridization signals were the most readily detected on interphase,the average value was 20.85%and 18.84%;pachytene took second place,10.26%and 11.36%;metaphase was the lowest,5.66%and 2.13%.Each probe picked up ten cells with visible hybridization signals and well-dispersed pachytene chromosomes were selected for measurement,the results showed the signals were located on the 16th chromosome.The relative length of the chromosome which contained signal of SSR(2301)was 3.475%,contained signal of SSR(2310)was 3.350%, contained two signals of SSR(2301)and SSR(2310)was 3.465%.Meanwhile,Image J was used to measure the location of each signals on the 16th chromosome and we found the location was 17.476±0.872%(SSR2301),44.911±0.029%(SSR2310).Signal location on metaphase was difficult to determine,for the chromosomes at this time shrank into granules with few disparities.