| Hevea brasiliensis is an important tropical crop and is also a special economic crop in China. There are many kinds of applications of molecular marker in the rubber tree,including RFLP,AFLP,RAPD,SSR,ISSR,EST-SSR, ILP,SNP and SRAP,which mainly being used in construction of genetic linkage map of rubber tree, species identification of rubber tree, molecular marker assisted breeding and genetic diversity research. 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. So this research started with a single chromosome,constructed the single chromosome DNA library of Hevea brasiliensis and obtained gene functional annotation after sequencing,aiming at providing a new perspective for the genome research of rubber tree, and the basis for molecular assisted breeding. The results of the study are as follows:?1? In this research, we used reddish bronze leaves of Hevea brasiliensis Reyan 7-33-97 as material to prepare the chromosome samples with the enzyme and low osmotic conditions, and 32 chromosomes were isolated from different metaphase cells. The 32 chromosomes were amplified by LA-PCR for two rounds, and 24 of them had banding.The main size ranged from 300 bp to 4000 bp. These 24 chromosomes were detected by Dot-blot hybridization to verify the source of the amplification, and 22 of them were hybridized successfully, it verified that the LA-PCR products of these 22 chromosomes were homogeneous with the Hevea brasiliensis genomic DNA. The 5 chromosomes which were verified successfully by Dot-blot hybridization were selected randomly to carry out Southern blot hybridization which further proved that they were derived from rubber tree.The 5 chromosomes were verified by fluorescence in situ hybridization combined with karyotype analysis, and then were identified as the third chromosome, the seventh chromosome, the thirteenth chromosome, the fifteenth chromosome and the sixteenth chromosome in Hevea brasiliensis Reyan 7-33-97, respectively.?2? The transformation system of the single chromosome library constructed was optimized. After optimization, the shaking incubation time was 45 min, the volume of the bacterial solution was determined to be 20?l, and the 37?inverted incubation time was 16 h. Then we constructed the microcloning libraries of chromosome 3, 7, 13, 15 and 16 of Hevea brasiliensis Reyan 7-33-97 successfully. The libraries of chromosome 3, 7, 13,15 and 16 contained 4.26×105, 3.6×105, 4.97×10O5,2.83×105 and 2.91×105 clones, respectively.In the microcloning library of chromosome 3, the main size of inserted fragments ranged from 250 bp to 850 bp, the average size was about 437 bp, and the library coverage rate was 2.1 times of the chromosome length. In the microcloning library of chromosome 7, the main size of inserted fragments ranged from 150 bp to 800 bp, the average size was about 382 bp, and the library coverage rate was 1.6 times of the chromosome length. In the microcloning library of chromosome 13, the main size of inserted fragments ranged from 250 bp to 1200 bp, the average size was about 491 bp, and the library coverage rate was 3.6 times of the chromosome length. In the microcloning library of chromosome 15, the main size of inserted fragments ranged from 250 bp to 800 bp, the average size was about 460 bp, and the library coverage rate was 2.1 times of the chromosome length. 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 456 bp, and the library coverage rate was 2.3 times of the chromosome length. The empty loading rates of five chromosome microcloning libraries were 3%, 7%, 2%, 1% and 2%, respectively.?3? The sequences of the positive clones in the libraries of chromosome 3, 7, 13, 15 and 16 of Reyan 7-33-97 with the numbers of sequences being 80, 88, 83, 81 and 111 respectively, were randomly sequenced. These sequences were aligned online with Reyan 7-33-97 whole genome database ?GCA001654055.1?, RRIM600 whole genome database?GCA 001907995.1?, EST database and the genetic map of rubber tree with 18 molecular markers on NCBI. Results showed that there were 19, 40, 15, 14 and 18 sequences having high homogeneous with the sequences of Reyan 7-33-97 whole genome database?GCA 001654055.1? in the five chromosome libraries; and there were 20, 42, 16, 29 and 21 sequences having more than 90% homology with Hevea brasiliensis EST sequences database, respectively, most of them were related to rubber tree cutting resistance, frost resistance, tapping panel dryness, salt resistance and the formation of latex after functional analysis. And the L-7Chr-4 was related to the formation of latex, and also had 97%homology with EST sequence GenBank: JZ378253.1, after functional annotation, the serial number was related to upland cotton seedling root resistance to salt stress.?4? The 21 sequences in the five chromosome libraries which had high homology with the whole genome database and EST database of Hevea brasiliensis were analyzed by Blast2GO. There were 14 sequences with high homology aligning successfully. The Blast2GO software was used to GO MAP matching, of which 8 sequences have been annotated with GO results. A total of 41 GO IDs were obtained, 22 of them were biological process, 12 of them were cellular component and 7 of them were molecular function. The 41 GO IDs were divided into 5 levels with different functions, in part of biological process, these sequences mainly participate in cellular process, metabolic process,single-organism process, biological regulation, biosynthetic process and primary metabolic process, and so on; In part of molecular function, these sequences mainly participate in catalytic activity, transporter activity and transferase activity, and so on; in part of cellular component, these sequences mainly participate in cell part, cell and organelle. Enzyme Code and KEGG metabolic pathway analysis were carried out after GO annotation, in library of chromosome 3, we obtained one Enzyme Code: EC2.7.7.49, but without result of metabolism pathways. |
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