Construction Of Five Chromosome DNA Libraries And Sequence Analysis In Hevea Brasiliensis Reyan 7-33-97

Hevea brasiliensis is the only cultivar among the trees of the Euphorbiaceae and Hevea tropical rain forests.It is also the most widely grown gum tree species in the world.The latex produced by the bark,which is used as a raw material for natural rubber.Therefore,rubber tree has a high economic value and strategic significance.At present,a series of molecular biological studies have been conducted about cDNA library and protein groups on the rubber tree,and got some progress.However,the related studies on the rubber tree about whole genome sequencing started relatively late.And the genome-wide sequencing of RRIM600,Reyan 7-33-97 and BPM 24 were completed in 2013,2016,and 2017,respectively.But these previous studies have only assembled to Scaffold levels and have not reached the chromosomal level.In this study,a rubber tree chromosome library was constructed and sequenced on a single chromosome level.Sequence annotation was used to functionally annotate some sequences of single chromosomes.The aim was to provide new ideas,new methods and basis of rubber tree molecular assisted breeding for rubber tree genome research at the single chromosome level.The results of the study are as follows:?1?In the experiment,we used the tender leaves of Hevea brailiensi'Reyan 7-33-97' as material to prepare chromosome samples with the enzyme and low osmotic methord.40 single chromosomes were isolated from the mid-stage cells of Hevea brasiliensis by means of the micro glass needle method,and used linker adaptor mediated PCR?LA-PCR?to randomly in vitro amplify the isolated 18 single chromosomes.After gel electrophoresis,18 bands were detected.The band size ranged from 200 to 4000 bp.Using Southern hybridization test verified 5 single chromosomes origin,which was selected at random among the 18 single chromosomes.The results showed that these 5 single chromosomes correspond with'Reyan 7-33-97' chromosomes of 5,6,11,13 and 14 respectively.?2?The above-mentioned 5 single chromosomes were amplified by LA-PCR for two rounds.And the purified products through ligated,transformed and cloned to obtain the 5 single-chromosomal micro-cloning libraries.In the constructed five chromosore libraries of 5,6,11,13,and 14,the number of clones were 4.5×105,5.11×105,3.32×100,3.01×10',and 4.41×105 in proper order,the size distribution of the inserted fragments were 250?1100bp,250?1200bp,300?1100bp,300?950bp,and 200?1100bp,the average length were 536 bp,481 bp,388 bp,401 bp,and 485 bp,and the coverage rates were reached 2.7,3.2,1.9,3.8 and 2.2 times,respectively.The 5 chromosome micro-cloning library empty loading rates were 2%,3%,3%,1%,2%respectively.?3?From the five clone libraries of chromosome 5,6,11,13 and 14 of,100 to 140 positive clones were randomly selected for sequencing.and was obtained.We compared sequencing information online with the Brazilian rubber tree of RRIM600 full genome database?GCA₀01907995.1?,'Reyan 7-33-97' genome database?GCA₀01654055.1?and the EST database in the NCBI.The alignment results showed that there were 23,30,21,21 and 18 sequences having high homogeneous with the sequences of Reyan 7-33-97 whole genome database?GCA₀01654055.1?in the five chromosome libraries;there were 23,28,22,20,and 18 sequences having high homogeneous with the sequences of RRIM600 whole genome database?GCA₀01907995.1?in the five chromosome libraries;and there were 20,74,40,38 and 57 sequences having more than 90%homogeneous with Hevea brasiliensis EST sequences database,Through the functional analysis,the function of the single chromosome sequences were mainly related to the salt tolerance,cut resistance,cut surface dryness,and frost resistance of the rubber tree.?4?The Blast2GO software was used to analyze the 19 homologous sequences,belonged to the five chromosomal libraries,which were highly homologous to Hevea brasiliensis and EST genome database.Seventeen sequences with high homology were found by comparison.GO MAP was performed on these 17 sequences to obtain terminology annotation results.A total of 23 GO term annotations were obtained,including 10 biological pathways,6 cellular components,and 7 molecular functions.The GO term annotations results were divided into 5 levels with different functions,which mainly involved in cellular processes,metabolic processes,single organism processes,biological regulation,biosynthesis processes and primary metabolism,etc.In terms of molecular function,it mainly participates in catalytic activity,transport activity and transferase activity,etc.We used Enzyme Code and KEGG metabolic to analyzed sequence after GO annotated and obtained Enzyme Code annotation on the 5th?6th?11th?13th and 14th chromosome libraries.In addition to the failure to annotate the function of related enzymes in the chromosome 11 library sequence,the Enzyme Code annotations were obtained in chromosome libraries 5th?6th?13th and 14th.And the L-5Chr-5,L-5Chr-91,and L-6Chr-48 sequences owned one Enzyme Code annotation.The annotated enzyme ID is 2.7.7.49,which is an RNA-guided DNA polymerase.In the 13th chromosome library,the sequence L-13Chr-25 owned an Enzyme Code annotation with the ID of 2.3.1.12,which is dihydrolipoyl lysine residual acetyltransferase.One Enzyme code annotation was obtained in the chromosome 14 library with the annotated enzyme ID of 3.6.1.15,which is a nucleoside triphosphate phosphatase,and the sequence was L-14Chr-73.However,the analysis of the KEGG metabolic pathway had no result in a metabolic pathway.Since the GO annotated sequence of the 11th chromosomal library failed to annotate the relevant enzyme function,it was not annotated sequences which participated in the related KEGG metabolic pathway.