| Rubber tree is the main source of natural rubber, and Hevea brasiliensis is an important species that produces latex at home and abroad currently. In-depth molecular cytology studies of Hevea brasiliensis are of great importance in the breeding of rubber trees. In recent years, researchers have completed the whole genome sequencing for three Hevea brasiliensis cultivars - RRIM600, Reyan 7-33-97, BPM24 - in 2013, 2016 and 2017 by using high-throughput sequencing technology. However, the genomic sequence assembly of rubber tree only reached the scaffold level and its genetic map, linkage group and other information does not matched to its chromosomes. Moreover, the position of the functional gene on the chromosome group and the linkage relationship have not fully determined yet. Hence, it is difficult to apply the results of the present research directly to the breeding of rubber trees. In this study, we constructed a single chromosome library of Hevea brasiliensis, which can solve this problem to a certain extent. Our research group first opened the construction of single chromosome library for rubber tree in China since 2013 and no other study in this research area has been reported. This study started from single chromosome,and 4 chromosomes of Hevea basiliensis were constructed by the technique of single chromosome microanalysis and LA-PCR amplification of single chromosome. The aim of this study was to provide a theoretical basis for molecular-assisted breeding of rubber tree. The main results for this study are as follows:(1) 31 single chromosomes were isolated from the multiple mesophase cells of Reyan 7-33-97 by using fine glass needle method and four of them were selected and amplified in two rounds of LA-PCR in vitro. The LA-PCR products were confirmed whether amplified from the genome sequence of Hevea brasiliensis via Dot-blot hybridization. The LA-PCR amplification products of the four chromosomes were labeled with DIG or BIO as probes and they were validated as belonging to chromosome 4, 12, 17 and 18 of Reyan 7-33-97 by fluorescence in situ hybridization (FISH) and karyotype analysis. The microclone library for the four chromosomes have been successfully constructed: the library of chromosome 4,12, 17 and 18 contains 2.11×105, 2.75×105, 2.77×105 and 1.32×105 clones, respectively; the size ranges of the inserted fragments were 250 ?1100 bp,250 ?1200 bp, 300 ?1400 bp,and 300 ?1100 bp with the average sizes were about 450 bp,500 bp,500 bp and 550 bp,respectively; the coverage rates of these 4 chromosomes were about 1.1 times, 1.9 times,2.4 times and 1.4 times, respectively, and the empty loading rates were 0%, 0%, 1% and 1%, respectively.(2) A total of 100 positive clones were randomly selected from chromosome 4, 12, 17 and 18 of Reyan 7-33-97. 100, 100, 101 and 99 sequences have been sequenced and 87, 83,91 and 87 uniq sequences were obtained after removing the same sequence for chromosome 4, 12, 17 and 18, respectively. These sequences were analyzed by the online BLAST software and 13, 11, 20 and 11 respective sequences from the chromosome 4, 12,17 and 18 were more than 90% homogeneous with the sequences of Hevea brasiliensis(RRIM600) whole genome database, respectively; and 17, 15, 23 and 13 sequences were more than 90% homogeneous with the sequences of Hevea brasiliensis (Reyan 7-33-97)whole genome database, respectively; and 18, 19, 19 and 14 sequences were more than 90% homogeneous with the sequences of Hevea brasiliensis EST sequences database,respectively. Most of the aligned EST sequence were related with the function of frost resistance, latex synthesis, rupture resistance and dead skin disease, etc., and are involved in the synthesis of amino acid precursors and LCR69 precursors and defensin protein precursors.(3) The Blast2GO software was used to analyze the 27 sequences from the 4 chromosomes with highly homologous with the rubber genome and EST database. The 8 sequences from NO.4 chromosome library produced 9 GO term annotations: 5 related molecular pathways (P), 3 related cell components (C), 1 related molecular function (F);The 7 sequences which in the NO. 12 chromosome library produced 9 GO term annotations:4 related molecular pathways (P), 3 related cell components (C), 2 related molecular function (F); the 6 sequences which in the NO. 17 chromosome library produced 8 GO term annotations: 3 related molecular pathways (P), 4 related cell components (C), 1 related molecular function (F); the 2 sequences which in the NO. 18 chromosome library produced 2 GO term annotations: 1 related molecular pathways (P), 1 related cell components (C),but without annotations of molecular function. The annotation of these sequences indicated that these sequences were involved in the process of cell proliferation, negatively regulated biological processes, cell regulation processes, biosynthesis, and N metabolism in biological pathways; In the molecular function, some sequences affect the activity of invertase; In cell components, these sequences are mainly involved in organelle composition, cell innate ingredients composition and other components. L-4Chr-25,L-12Chr-59 and L-17Chr-4 in the chromosome library of chromosome 4, and 17,respectively, obtained the same enzyme annotation: glutamine adenosine transferase (EC 2.7.7.49), KEGG metabolism pathway analysis, no metabolic pathway. |
PDF Full Text Request