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

Hevea brasiliensis is an important rubber producing plant,which has the advantages of long life,high yield and easy obtaining latex.At present,molecular markers are widely applied in the identification,genetic map,breeding and genetic diversity analysis of Hevea brasiliensis.But the genetic map,which has been reported,is few,and it has some disadvantages such as linkage instability,small abundance and uneven distribution.The whole genome sequencing of Hevea brasiliensis was carried out later,and the whole genome sequencing of RRIM600,Reyan 7-33-97 and BPM 24 were completed in 2013,2016,and 2017,respectively.But the assembly level only reached the level of scaffold,which do not reach the level of chromosome.In this study,we constructed the single chromosome DNA library of Hevea brasiliensis by the micro glass needle method,and analyzed the gene alignment and gene annotation of the part of the libraries.The aim is to provide a new auxiliary method for the study of the genome of rubber tree and provide the basis for the molecular assisted breeding of rubber tree.The results of the study are as follows:?1?We used the tender leaves of Reyan 7-33-97,an improved method was used for the enzyme and low osmotic method.The results showed that when phosphate buffered saline?NaH2PO4·2H2O,29.18 g/L;Na2HPO4·12H2O,4.66 g/L;pH=5.5?was used as solvent and dissolved enzyme mixture?5%cellulase and 4%pectinase?,the time of enzymatic hydrolysis was 1.0?1.5 h at 37?.?2?We used the efficient technique of chromosome glass needles microdissection,7 single chromosomes were successfully isolated from the metaphase cells of Reyan 7-33-97.The products were verified by Southern blot that the LA-PCR products were homogeneous with the genomic DNA of the rubber tree.A total of six chromosomes were verified by FISH and Karyogram,and then were identified as the first chromosome,the second chromosome,the third chromosome,the fifth chromosome,the thirteenth chromosome,the Sixteenth chromosome in Reyan 7-33-97.?3?Then we constructed the microcloning libraries of chromosome 1,2,3,5,13 and 16 of Reyan 7-33-97 successfully.The libraries of chromosome 1,2,3,5,13 and 16 were contained 4.26×105,3.6×105,4.97×105,4.97×105,2.83×105 and 2.91×105 clones,respectively.In the microcloning library of chromosome 1,the main size of inserted fragments ranged from 200 bp to 1000 bp,the average size was about 398 bp,and the library coverage rate was 1.0 times.In the microcloning library of chromosome 2,the main size of inserted fragments ranged from 300 bp to 1000 bp,the average size was about 396 bp,and the library coverage rate was 1.2 times.In the microcloning library of chromosome 3,the main size of inserted fragments ranged from 200 bp to 1000 bp,the average size was about 451 bp,and the library coverage rate was 1.9 times.In the microcloning library of chromosome 5,the main size of inserted fragments ranged from 200 bp to 1000 bp,the average size was about 455 bp,and the library coverage rate was 1.5 times.In the microcloning library of chromosome 13,the main size of inserted fragments ranged from 250 bp to 1000 bp,the average size was about 447 bp,and the library coverage rate was 1.9 times.In the microcloning library of chromosome 16,the main size of inserted fragments ranged from 250 bp to 1000 bp,the average size was about 493 bp,and the library coverage rate was 2.6 times.The empty loading rates of six chromosome libraries were 2%,2%,1%,3%,2%and 3%,respectively.?4?The part of sequences of the positive clones of chromosome 1,2,3,5,13 and 16 of Reyan 7-33-97 were obtained at random,and the numbers of sequences were 172?166?114?106?83and 89,respectively.These sequences were aligned with Reyan 7-33-97?GCA₀01654055.1?,RRIM600?GCA₀01907995.1?,EST database on NCBI.Results showed that there were 23?45?45?53?25 and 28 sequences having high homogeneous with the sequences of Reyan 7-33-97?GCA₀01654055.1?in the six chromosome libraries;and there were 18?50?29?34?42 and 20 sequences having more than 90%homogeneous with EST sequences database of Hevea brasiliensis,respectively,most of them were related to cutting resistance,frost resistance,tapping panel dryness,salt resistance and the formation of latex after functional analysis.?5?The 28 sequences in the six chromosome libraries were analyzed by Blast2GO,which had high homologous with the whole genome database,there were 15 sequences having GO annotations.A total of 50 GO IDs were obtained,18 of them were biological process,14 of them were cellular component and 18 of them were molecular function.In the part of biological process,these sequences were mainly participating in cellular process,metabolic process,single organism process,biological regulation and biosynthetic process,and so on;In the part of molecular function,these sequences were mainly participating in catalytic activity,transporter activity and transferase activity,and so on;in the part of cellular component,these sequences were mainly participating in cell part,cell and organelle.Enzyme Code and KEGG metabolic pathway analysis were carried out after GO annotation,in the library of chromosome 1,we obtained two Enzyme Code:EC 2.7.7.49,EC 3.4.23,in the library of chromosome 2,we obtained one Enzyme Code:EC 3.2.1.23,but with no result of all the metabolism pathways.