| Mulberry is a perennial economic tree belonging to Moraceae,Morus.Apart from cultivation for feeding silkworm,mulberry still have high edible and medicinal value.Mulberry plays a certain role in wind and sand fixation and ecological management because of its stress resistance.Chromosome studies are of great reference value in the analyses of the origin,phylogeny,and phylogenetic relationship of species.Most chromosome studies of mulberry focused on the chromosome counting and ploidy analyses,lacking systematic karyotype analyses.The main reason is that the chromosomes of mulberry are all dot chromosomes and hard to prepare,except Morus notabilis and M.yunnanensis.Several studies have applied the fluorescence in situ hybridization technique in mulberry.However,these studies cannot identify all the chromosomes and perform systemic karyotype analyses,which slow the chromosome study of mulberry.The genome sequencing of M.notabilis provides the platform for us to analysis chromosome in molecular level.In this study,single copy sequences and rDNA sequences from M.notabilis were selected as probes,and systemic chromosome identifications of mitotic and diakinesis chromosomes were performed.Based on the relative length of diakinesis chromosomes,all the chromosomes were correctly ordered and the kayotypes of M.notabilis were constructed.The mitotic chromosome complement of M.notabilis is different with meiotic chromosome complement,indicating a chromosomal fusion-fission cycle in M.notabilis.Further,the cycle is widespread in mulberry.The cycle involved diakinesis chromosome 1 of lunjiao109 was microdissected.Diakinesis chromosome 1 sepecific DNA libraly was constructed to select the elements involved in chromosomal fusion-fission cycle and to illustrate the mechanism.The main results are as follows:1.Mitotic and diakinesis karyotypes of M.notabilisSingle copy sequences with the length of >10kb were selected based on the BLASTn searches against the genomic database of M.notabilis.Four single copy sequence probes(morus027496,morus027717,morus026579,and SSR2524)were shown in this study.The 5S and 25 S rDNA reported previously were also used in this study.Mitotic chromosomes and diakinesis chromosomes were used in the chromosome identification using fluorescence in situ hybridization.All the chromosomes were located by one probe.Chromosome 1 and 4 were easily identified by the morphology.Chromosome 2 and 3 were distinguished from the different location patterns of probes.Three dot chromosomes were also distinguished and ordered from the differences in signals and chromosome length.More morphology characteristics were showed in surprising six bivalents in diakinesis phase.Distinct primary constriction was occured in diakinesis chromosome 3.Diakinesis chromosome 5 was shown special morphology with small short arm and large long arm.Based on the chromosome morphology,signal patterns,and relative length of diakinesis chromosome,all the mitotic chromosomes and diakinesis chromosomes were ordered.Finally,mitotic and diakinesis karyotypes of M.notabilis were constructed.2.Chromosomal fusion-fission cycle in mulberryBased on the karyotype analyses of M.notabilis,different number was shown between mitotic chromosome complement and diakinesis chromosome complement.Probe 25 S rDNA was used to trace the alteration of chromosome number in mitotic and meiotic phases.During the meiotic phases,all the chromosome 5 and 7 were separated in leptotene phase;homologous chromosomes were synapsised during zygotene,the number of 25 S rDNA signals were accordingly reduced to two;Homologous chromosomes synapsised completed during pachytene phase,the number of signal turned to one;During the later phases,the signals of 25 S rDNA were separated following the separation of chromosomes,and no chromosome lag and tails were shown.In conclusion,chromosome 5 and 7 were fused during the phases from leptotene to pachytene.11~20% cells containing 12 chromosomes were shown in all of the three adult tree of M.notabilis,most of the cells were had 14 chromosomes.The result suggested that fused chromosomes were broken in the plant.Repeated chromosome fusion and fission implied the unique chromosomal fusion-fission cycle in M.notabilis.Chromosome 5 and 7 were fused between leptotene phase and pachytene,the fission process was happened in the whole life cycle.25S rDNA were located on the chromosomes of polyploid mulberry.The results showed that 2,3,and 4 loci were found in the mitotic cells of tetraploid mulberry,only one locus in the diakinesis and pachytene chromosomes.The location pattern was the same with M.notabilis.Variable loci of signals were also found in higher ploidy level mulberries.Location pattern of 25 S rDNA in polyploid mulberry was consistent with M.notabilis.The signals were located in the middle of the large dot chromosomes or the distal part of dot chromosomes.In conclusion,the chromosomal fusion-fission cycle is widespread in mulberry.3.Chromosomal microdissection in mulberry and the mechanism of chromosomal fusion-fission cycleThe chromosomal fusion-fission cycle involed diakinesis chromosome 5 is similar with diakinesis chromosome 4 M.notabilis.Diakinesis chromosome 1 is the cycle involed chromosome in polyploid mulberry,which is easier to be distinguished.Tetraploid mulberry contained relative smaller chromosome number.Thus,diakinesis chromosome 1 of lunjiao109(2n=28)were selected to illustrate the mechanism of fusion-fission cycle in mulberry.Fifty diakinesis chromosome 1 were microdissected here.After amplified using single cell amplication kit,diakinesis chromosome 1 specific DNA library was constructed.Twenty-one clones with repeat sequences were selected through dot plot.These clones showed 4 type signal distribution patterns,which are the whole chromosome distribution signals,chromosome end localization signals,partial chromosome distribution signals,and dot signals.So far,no sequence with signal distribution at the fusion and fission site has been obtained,and the work of library screening is still underway.In this study,systemic mitotic and diakinesis karyotypes of M.notabilis were constructed using fluorescence in situ hybridization,laying a solid foundation for the chromosome study of mulberry trees.Dynamic changes of chromosomes in various stages of mitosis and meiosis were analysed,proposing the chromosome evolution model in M.notabilis was chromosomal fusion-fission cycle.It is the first time to be reported that chromosomal fusion-fission cycle exists in the process of generation alternation within an individual.Further,the cycle is widespread in mulberry.The application of chromosomal microdissection technology provides a platform for studying the structure of single chromosomes of mulberry trees.The selected sequences provide a variety of probe candidates for mulberry chromosome research.In this study,systemic mitotic and diakinesis karyotypes of M.notabilis were constructed,and chromosomal fusion-fission cycle was proposed as the chromosomal evolution model of mulberry,providing important references for the studies of origin and evolution of mulberry trees. |
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