Screening Of Chromosomal Marker And Chromosome Identification In Erianthus Arundinaceus

Sugarcane(Saccharum spp.)is a most important sugar crop in China,however,its narrow genetic background had hindered the progress of sugarcane breeding.Erianthus arundinaceus,as a related wild genus of sugarcane,has many excellent traits for sugarcane genetic breeding.While,the noblization hybridization process of E.arundinaceus is relatively slow,because a specific inheritance of chromosome doubling was found in the BC1 progeny between sugarcane and E.arundinaceus.In order to understand and definite the mechanism of chromosome inheritance in noblization,the following study were carried out.Firstly,genomic DNA of E.arundinaceus was broken,and a technique system for Cot enrichment of middle and high copy repeat sequences was established by Reverse Dot Blotting(RDB).Secondly,repeated sequences were used as probes to locate on the chromosomes of E.arundinaceus by fluorescence in situ hybridization(FISH),and obtaining different types of probe sequences.Thirdly,E.arundinaceus karyotypes were constructed by using mid-high copy repeated sequences and low copy BAC sequences.The main findings are as follow:1.Constructing a Cot DNA library:The genomic DNA was randomly interrupted and four renaturation time gradients of Cot-1,Cot-20,Cot-60,and Cot-100 were seted.We verified by RDB technique that enriched middle-copy sequences of Cot-20 were 31.25%,and the high-copy sequences accounted for 16.67%.At the same time,the diversity of signal sites of middle-copy sequences on the chromosome of E.arundinaceus was verified by FISH,which is benefitted to screen differentially probes.Hence,we used Cot-20 as renaturation time for constructing Cot library.A Cot DNA library containing medium and high copy probe sequences was obtained.2.Analysis of different repeated sequences located on chromosome:the repetitive sequences were obtained from the Cot library and used to locate on the metaphase chromosome of E.arundinaceus by FI SH.The 1350 probes were screened and could be divided into 9 types;the number of subtelomere at both ends was 727,53.85%of the total probes;One-end Subtelomere was 163,12.07%of the total probes;subtelomere at one end and both ends was 31,about 2.30%of the total probes;A small number of centromeres and subtelomere at both ends was 23,1.70%of the total probes;Subtelomere at both ends and centromeres was 38,approximately 2.81%of the total probes;The central dispersions was 6,0.44%of the total number.There are 25 centromeres,1.85%of total probe;51 spread,3.78%of the total probes,and 286 with no signal,21.19%of screened probes.3.Perfected the technique of repeated FISH on E.arundinaceus:The previously hybridized probes can be completely washed out without any affection in the next hybridization when the chromosome slides that were previously hybridized had been eluted with different intensities.At present,it is proved that this hybridization can be repeated three times in our laboratory.Repeated hybridization can effectively determine whether the probes are the same type and draw chromosome karyotypes on the same metaphase chromosome,and can also save chromosome samples.4.The chromosome karyotype and chromosome recognition system of E.arundinacea were preliminarily constructed:According to position and color differences,different types of probes were used to locate on the chromosome of E.arundinaceus.After multiple combinations and hybridizations,we finally identification 8 chromosome karyotypes using probes Ea-0907,Ea-0098,and 45S rDNA;at the same time,using seven different probes,we constructed the FISH fingerprinting map of E.arundinaceus chromosomes through three repeated hybridization.5.The evolution of E.arundinaceus,sorghum and sugarcane was analyzed by using sugarcane low copy BAC:The cognate relation of 27 low-copy BAC sequences were analyzed in sugarcane,E.arundinaceus and sorghum by using RDB.And then the homology and collinearity of these sequences in different species during the evolutionary process were initially determined.Finally,the results were further verified by FISH.However,there were only one BAC-2 sequence in 27 sugarcane BAC sequences with high homology in all tested materials.