Cytogenetic Anlysis Of Six Rubus L. Species By Meiosis And FISH

Southeast China is considered to be a major center of diversity and provide an abundant source of previously untapped germplasm for breeders, consisting about200and with complex types, ploidy of variation chromosomes, from herbaceous to woody species. However, few germplasm resources were utilized for there was almost no deep study on them. Therefore, systematic taxonomy, genetic relationship, and origination of Rubus L. were always of great importance for improve their characteristic. Recently, many techeniques, such as morphology, chromosome karyotype, palynology, DNA molecular etc. were used to study on these ways, but there is little about the meiosis and FISH analysis.In this paper, six excellent germplasm Rubus L. species (R. ellipticus var. obcordatus, R. coreanus, R. assamensis, R. lambertianus var. glaber, R. setchuenensis and R. buergeri) were analyzed, through meiosis study and rDNA-FISH to observe the chromomsome composition and rearrangement, the distribution characteristics of rDNA. The meiosis study and rDNA-FISH were carried out to investigate and analysis their phylogenesis, genetic relationship and formation polyploidy. And the results would be list as follows:(1) The meiotic pairing at M Ⅰ of six excellent germplasm Rubus L. species were analyzed, the results showed that the R. ellipticus var. obcordatus with no bivalent were observed in M I, which is different to R. coreanus Miq. and the average chromosome pairing configuration were6.98Ⅱ+0.02Ⅰ,7Ⅱ, respectively; meiotic pariring in the three tetraploids of R. assamensis, R. lambertianus Ser. var. glaber, and R. setchuenensis (2n=4x=28) were13.7Ⅱ+0.3Ⅰ,13.1Ⅱ+0.9Ⅰ and13.4Ⅱ+0.6Ⅰ; the chromosome configuration of R. buergeri known to be octoploid (2n=8x=56) were1.89Ⅰ+23.2Ⅱ+0.116Ⅲ+0.18Ⅳ per cell. Abnormalities of meiosis in Rubus such as chromosome largging and micronuclei were investigated, and with the increase in chromome, the number of lagging chromosomes is also rise. These partly implied that the Rubus L. were polyploidy evolution. The R. assamensis, R. lambertianus Ser. var. glaber, R. setchuenensis and R. buergeri were alloployploids.(2) Synthesize the primer of5S rDNA nucleotide sequence, deal with the dNTP, concentration of primers, Mg2+and so on by orthogonal design and optimization. The results showed that the optimal PCR amplification system were as follows:20ng Rubus DNA template,0.2μM each Primer,0.2mM dNTP,2mM Mg2+,2.5uTaq DNA enzyme,5μL PCR Buffer.(3) Prior to hybridization, a series of pre-treatment were utilized to reduce the packages of cell wall and cytoplasm for enhancing the penetration of cells and declining the signal background. And the suitable time of the enzyme mixture with2%pectinase and2%cellulose is1.5h at37℃.(4) Fluorescent in situ hybridization (FISH) was carried out to analysis the distribution patterns of the45S rDNA and5S rDNA on chromosomes of six species in Rubus L.. The results showed that the rDNA sites were variability in the number, size, and location of the two diploids:there were both two45S rDNA and5S rDNA in R. ellipticus var. obcordatus which were all found on the satellites of chromosome Ⅶ; in R. coreanus, four45S rDNA sites, which were found in the terminal regions of chromosome Ⅱ and Ⅳ, and two5S rDNA sites, which were found close to centromere on the chromosome5were detectd. During the ployploids, four45S rDNA and two5S rDNA signals were both detected in R. assamensis and R. lambertianus var. glaber, while only three45S rDNA and two5S rDNA were found in R. setchuenensis. In R. buergeri, there were eight45S rDNA and three5S rDNA. These results implied that45S rDNA and5S rDNA sites were all localized on the terminal regions on the chromosome arms and through the intensity, the size and number of rDNA sites, we can further improve that Rubus L. were polyploidy evolution and the R. assamensis, R. lambertianus Ser. var. glaber, R. setchuenensis and R. buergeri were alloployploids.