Construction Of Three Chromosoma DNA Libraries And Preliminary Analysis Of High Throughput Sequencing Of The Fourth Chromosome In Hevea Brasiliensis Reyan-7-33-97

Hevea brasiliensis,a member of the Euphorbiaceae family,is a kind of natural rubber-producing plant and an important economic crop.At present,the applications of molecular marker technology in rubber research have gotten certain progress,but the molecular biology research is still deficient.The saturation is not enough about these constructed genetic maps of rubber tree,and many genes location and linkage on chromosoma are also unable to be confirmed and it is also difficult to be used for rubber tree breeding research directly.These problems can be resolved effectively by starting with a single chromosome,and constructing the single chromosome DNA library of Hevea brasiliensis and obtaining gene functional annotation after high-throughput sequencing.Related researches about the rubber tree libraries were from the whole genome or cDNA libraries.However,the related research has been reported rarely expect that our team have carried out research on Hevea brasiliensis single chromosome library construction since 2013.In respect of rubber tree high-throughput sequencing researches,only the draft genome sequence of the RRIM600 rubber tree and the RNA-seq of other rubber tree varieties were reported,the research on Hevea brasiliensis single chromosome high-throughput sequencing was rare.In this thesis,single chromosome microdissection technology was used to separate the single chromosomes of rubbertree "Reyan 7-33-97".We constructed the single chromosome microcloning library after single chromosome amplificated by linker adaptor mediated PCR(LA-PCR).Moreover,we used Illumina platform for high-throughput sequencing after the single chromosome amplificated by single-cell whole genome amplification method.It will provide a new perspective for the study of rubber tree genome and a molecular assisting way for breeding of rubber tree.The results of the study are as follows:(1)In this study,we used tender leaves of Hevea brasiliensis Reyan-7-33-97 as material to prepare the chromosome samples with the enzyme and low osmotic method.It would gain a high quality chromosome samples by enzymd for 7h40min and low osmoticed for 30min.Using the efficient technique of Hevea brasiliensis chromosome glass needles microdissection that established by laboratory other members,29 single chromosoma were successfully isolated from different metaphase cells of Reyan 7-33-97.Three randomly selected chromosoma were amplified by linker adaptor mediated PCR(LA-PCR)for two rounds.The products were verified by Southern blot hybridization that the LA-PCR products were homogeneous with the Hevea brasiliensis genomic DNA.The result showed that these chromosoma had been successfully amplified.Two rounds LA-PCR products of the three chromosoma were marked as probes by DIG.Then the three chromosomes number were verified by fluorescence in situ hybridization(FISH)and Karyogram.The chromosoma were the tenth chromosome,the eleventh chromosome and the fourteenth chromosome in Hevea brasiliensis Reyan 7-33-97.(2)We constructed the three chromosoma microcloning libraries of Hevea brasiliensis Reyan 7-33-97.The librariy of chromosome 10?11 and 14 respectively contains 1.8×105,2.4×105 and 1.5×105 clones.After PCR assaying the positiveclones,in the microcloning library of chromosome 10,the main size of inserted fragments ranged from 200 bp to 1100 bp,and average size was about 400 bp,and the library coverage rate reached 7 times of the chromosome length.In the microcloning library of chromosome 11,the main size of inserted fragments ranged from 200 bp to 1100 bp,and averaged size was about 450 bp,and the library coverage rate reached 5 times of the chromosome length.In the microcloning library of chromosome 14,the main size of insertsed fragments range from 300 bp to 1100 bp,the averaged size was about 450 bp,and the library coverage rate reached 5 time of the chromosome length.Further measurement we learned that the three chromosoma microcloning library empty loading rate were 5%,4%and 5%,respectively.(3)One hundred random positive clones respectively from the library of chromosome 10,11 and 14 were sequenced.A total of 100,100 and 94 sequences were obtained and there were 93,91 and 86 sequences after removing duplicates in the three chromosome library,respectively.These sequences were performed by BLAST online.Results showed that there were 1,3 and 3 sequences more than 90%homogeneous with the sequences of Hevea brasiliensis WGS(RR-IM600)database in the three chromosome library,and there were 1,3 and 4 sequences more than 90%homogeneous with Hevea brasiliensis EST sequences database,respectively.(4)These sequences in the three chromosome library that high homologous with rubber tree genome database and EST database were analyzed by Blast2GO.In the tenth chromosome library,there are two sequences analyzed by Blast2GO software,and produced seven GO term annotations as a result.There were two GO term annotations about biological pathways and five GO term annotations about the molecular function;Enzyme Code and KEGG metabolic pathway analysis with obtained GO annotation sequences of chromosome 10 libraries showed that there were 2 Enzyme Code,EC3.6.1 and EC3.6.15,participating in the metabolism of purine metabolism and thiamine metabolism pathways.In the eleventh chromosome library,there were 6 sequences analysised by Blast2GO software,and produced 0 GO terms annotation.There were only 2 sequences with 8 GO Mapping annotation result.There were three GO term annotations about biological pathways and three GO term annotations about cellular component and two GO term annotations about the molecular function.In the fourteenth chromosome library,there were seven sequences analyzed by Blast2GO software,and produced 11 GO term annotations as a result.There were five GO term annotations about biological pathways and cellular component,and one GO term annotations about the molecular function.Enzyme Code and KEGG metabolic pathway analysis with obtained GO annotation sequences of chromosome 14 libraries indicated that there were two same Enzyme Code EC2.7.7.49,but without result of metabolism pathways.(5)A chromosome was amplified by single cell and genome PCR for two rounds.The products were verified by Southern blot hybridization and the PCR products were homogeneous with the Hevea brasiliensis genomic DNA.The chromosome was verified by fluorescence in situ hybridization(FISH)and Karyogram.It was the fourth chromosome in Hevea brasiliensis Reyan 7-33-97.(6)The PCR products of chromosome 4 were sequenced by high-throughput in Illumina platform.After double filtration of sequence results,we got 770470 reads with the size of 100 bps mapped to the genome sequence.By the mutation detection we got 8019 SNP variations,82 InDel sites,and 184 SV sites.(7)After triple filtration of sequence results,we randomly selected 2000 sequences to blast with(RRIM600)WGS database.There were 1846 sequences reached more than 99%homology.78%of these sequences were of the high sequence homology with assembly WGS database and others were of the high sequence homology with unput WGS database.We randomly selected 3000 sequence to blast with EST database.There were 25 sequences with more than 90%homology.24 sequences of the high homology with EST database were selected to be analyzed by Blast2GO.In the fourth chromosome,there were five GO term annotations as a result.There was one GO term annotations about biological pathways and cellular component repectively,and three GO term annotations about the molecular function.Enzyme Code and KEGG metabolic pathway analysis with obtained GO annotation sequences of chromosome 4 libraries demonstrated that there were no Enzyme Code and no metabolism pathways.