Microdissection Of Three Chromosomes And Preliminary Analysis Of High-throughput Sequencing Of The Tenth Chromosome In Cassava

Cassava(Manihot-esculenta Crantz)has biological characteristics such as high starch yield,high photosynthetic efficiency and drought tolerance.Since Cassava roots are rich in starch,cassava is not only a kind of important tropical food crops in Euphorbiaceae family,but also the ideal biomaterial for production of renewable bio-fuel.Therefore,it is of great significance to make the intensive study on cassava genome.Domestic and international researches on cassava genome had been very in-depth,but there were some shortcomings in researches of which KU50.W14 and AM560 were whole-genome sequencing,no genetic linkage group classification,nor located in the corresponding chromosome.Cassava is a cross pollinated plant.It has a highly heterozygous genome for the years of asexual reproduction,existing in the whole genome sequencing could not distinguish the DNA sequence of homologous chromosomes.To deal with the above problems,this study intends to use tiny glass needle microdissection method for cutting the single chromosome of SC6.After making the targeted chromosome amplification,and then we carried out high-throughput sequencing.Cassava single chromosome.microdissection and sequencing,can provide useful basis of molecular genetics for perfecting the existing cassava genome project,and can provide effective molecular assisted breeding of single chromosome specific probe and molecular markers.The specific research results are as follows:(1)In this study,we used apical root of SC6(2n=36)as material to prepare the chromosome specimens with the enzyme and low osmotic method.Pretreatment method of cassava apical was optimized,and the material pretreatment way for separating chromosomes of SC6 was established preliminarily.(2)25 chromosomes were successfully isolated from different metaphase cells of SC6 by using the method of slender glass needle(Part of the chromosome has been separated repeatedly),and there were 5 chromosomes from one metaphase cell of SC6.(3)We made improvement on key operations in FISH,such as the rinsing time and preliminarily established an efficient technique of FISH;The method that reusing the cassava chromosome specimens in the process of FISH was foud.(4)Three chromosomes were amplified by MALBAC for two rounds.Production was verified by Southern blot hybridization,results showed that the MALBAC products were homogeneous with the cassava genomic DNA,indicating that DNAs from the chromosome have been successfully amplified.The three chromosomes were verified by FISH and karyogram.They are the ninth chromosome and the tenth chromosome and the thirteenth chromosome in SC6.(5)The MALBAC PCR products of the chromosome 10 were tested with Illumina platform by using high-throughput sequencing technology,we obtained 3,316,798 valid sequences which reads were 125 bp long and 84,003 of them were homogeneous with the sequences of AM560,at a rate of 2.53%.After variation detecting,we obtained 401 SNP loci and 8 InDel,and these mutation detections were coupled structural and functional annotation,then the sequencing results were spliced together.(6)Having compared the 251 assembled contigs respectively with three whole genome segments of KU50,W14,AM560,the comparison of the results showed that there were 7 sequences reached more than 90%homology with KU50,5 sequences reached more than 90%homology with W14 and 7 sequences reached more than 90%homology with AM560.