Construction And Application Of New Chromosome Recognition System For Sugarcane

As the main raw material of sucrose,sugarcane(Saccharumspp.)is the most important sugar crop in the world.However,the number of chromosomes in sugarcane is large and different,which makes it difficult to carry out the genetic evolution and diversity of sugarcane genome.Oligo-FISH is a new molecular cell genetic technology,according to the genome sequence,design and synthesize specific sequence probe,chromosome and chromosome segments can be accurately identified.Especially for species with large number of chromosomes and high repeat sequences.Compared with traditional cytology and conventional genomic methods,it has more advantages in the study of the structure and evolution of chromosome groups and the application of chromosome engineering breeding.This study is based on Oligo-FISH technology,for the important wild species of sugarcane,a chromosome specific Oligo probe was successfully developed and it laid a foundation for the subsequent studies on sugarcane genome.The main research contents are as follows:1.The preparation of sugarcane oligo probe and the establishment of Oligo-FISH technology system:This study is based on high homology of sugarcane and sorghum,based on the sequenced sorghum genome sequence,through sequence comparison analysis,remove repeated sequences and design oligo pool and synthesize,the synthesized oligo library was amplified by emulsifying PCR,in vitro transcription,reverse transcription and enzymatic hydrolysis,the single stranded oligo probe with the tag is obtained,the single strand oligo probe with the tag is obtained,A clear FISH signal can be obtained from the experience certificate.The Oligo-FISH technology system was successfully established,the results showed a high similarity between homologous chromosomes.2.Screening of SES208 chromosomal specific marker probe:Fl-uorescence in situ hybridization was used to verify the cytology of 20 oligo probes,through the observation and analysis of the late s ignal,the probe with no main signal and high background interfere nce was removed,the seven specific oligo probes that can accuratel y identify the Saccharum spontaneum L.SES208 1-7 chromosome were selected.The results showed a high similarity between homolo gous chromosomes,this is the hypothesis of the homologous octadi ploid.3.The chromosome localization of 45 S rDNA and 5S rDNA locus was analyzed in the chromosomal localization of Saccharum spontaneum L.SES208:In this study,the 45 S rDNA and 5S rDNA were prepared by the gap translation method.Combined with the obtained specific probe of Saccharum spontaneum L.SES208 1-7 chromosome,cytology test by multicolor FISH technology,The experimental results showed that the 5S rDNA had 8 sites and 45 S rDNA had only 7 sites on the chromosome of Saccharum spontaneum L.SES208,the 5S rDNA was located on chromosome 7 of Saccharum spontaneum L.SES208,and 45 S rDNA was located on chromosome 5 of Saccharum spontaneum L.SES208.The development of chromosomal specific probe and Oligo-FISH technology can be used to establish the specific identification of sugarcane chromosome,it provides a very powerful cytological tool for the study of chromosome composition and evolution of sugarcane,especially for Saccharum spontaneum L.,It will also lay a foundation for the effective utilization of the Saccharum spontaneum L.and the related research on genetic evolution of sugarcane.