| Sugarcane(Saccharum spp.)is the major sugar crop and renewable bioenergy crop,narrow genetic has restricted the improvement of the yield and quality traits.Erianthus arundinaceus is an important wild germplasm resource of sugarcane with many excellent traits which could be used in sugarcane breeding program.However,the utilization process of E.arundinaceus is relatively slow,because the mechanism of chromosome inheritance in the noblization process is unclear.In order to investigate the mechanism of chromosome inheritance in the noblization process,it is an essential study to screen out the specific chromosome marker.Therefore,the following study was carried out.Firstly,the optimization system of suppression subtractive hybridization(SSH)technology was built based on the genomic DNA of E.arundinaceus and sugarcane,and then the efficiency was verified by reverse dot blot(RDB).Secondly,specific primers were designed by the sequences from subtractive library,which could be used to detect the authenticity of progeny between E.arundinaceus and sugarcane.Finally,the specific sequences as probes from subtractive library were used to perform physical map on chromosomes of E.arundinaceus for establishing chromosome karyotype of E.arundinaceus and investigating its chromosome inheritance in progeny between E.arundinaceus and sugarcane.The main results were as follows:1.The SSH optimization system was built in E.arundinaceus and sugarcane:0.1-1.5 Kb fragment can be achieved by double enzyme digestion;150 ng/μL double digested production was ligated with Alu I ligation and its efficiency was the highest.During the process of nested PCR,increasing the usage of primers and polymerase could ensure the fragment be effectively amplified in the second PCR.The results of substractive efficiency showed that the optimization system of the substractive library had high efficiency.2.The subtractive library was obtained with high efficiency:135 positive fragments were obtained from subtractive library of E.arundinaceus,and the positive rate was 71.35%,the length of sequences was varied from 60 to 894 bp;164 positive fragments were obtained from sugarcane subtractive library,varying from 150-1000 bp,and the positive rate was 85.42%.The RDB results showed that specific positive rate was 82.69%from subtractive library of E.arundinaceus and 89.58%from subtractive library of sugarcane,suggesting that the forward and reverse substractive librarys had high efficiency.3.The specificity of substractive library was verified:Specific primers were designed by two sequences obtained from E.arundinaceus and sugarcane substractive library,respectively.Results showed that Bm097166 and Bm020-127 could be only amplified in five materials from E.arundinaceus materials,Gz356 and Gz264 could be only amplified in thirty-one sugarcane materials,indicating that the positive clones were specific in other materials,which could provide effective and specific markers to identify the authenticity of progeny between E.arundinaceus and sugarcane.4.A large number of repetitive sequences were obtained by FISH for establishing chromosome karyotype of E.arundinaceus:In the subtractive library of E.arundinaceus,four positive clones were ramdomly selected as probes to perform FISH mapping on chromosomes of Yacheng 96-40,a F1 progeny between E.arundinaceus and sugarcane.Results showed that only E.arundinaceus chromosomes had obvious signals,which was consistent with the RDB result.By using the remaining 82 specific probes,FISH was conducted in Hainan 92-77.Results showed that 54 probes had signals:48 probes were in telomeric region,6 in centromeric region;the ratio of repeat sequences was 62.49%.It showed that a lot of repeat sequences could be obtained from the optimization system of the substractive library of E.arundinaceus,which could be used for chromosome karyotype analysis in E.arundinaceus,and could be used to track the chromosome inheritance of progeny between E.arundinaceus and sugarcane. |
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