Studies On The Chromosome Ploidy And FISH Analyses Of Mulberry Plants(Morus L.)

Mulberry is a perennial woody plant belonging to Moraceae, Rosales, and also is an important ecological and economic tree. For a long time, the studies on mulberry have been mainly focused on germplasm resources, disease and pest control, cultivation and propagation. Results on mulberry cytologyic studies have been reported a lot, but most of them were on the chromosome number, not reach to the karyotype analysis. Studies of mulberry chromosomes can provide not only the cytological evidences for the classification, origin, evolution, phytogeny and cell heredity, but also the ploidy, the fertility and the genetic relationship of parent materials in breeding. Since the chromosomes of mulberry plants are very small and the numbers are very large, it is quite difficult to prepare chromosome section with high quality using tranditional method and thus limited the cytology research of mulberry plants. The research in the field of molecular cytology of mulberry plants has not been reported yet.Fluorescence in situ hybridization (FISH) is based on the principle of complementarity, which can be used in obtaining the precise position of modified nucleotide probe on chromosomes, and could offer recognized stamps for chromosomes. FISH is simpler, intuitive, faster, and more accurate compared to the traditional methods. This technology can locate the repetitive sequences on chromosomes and study their distributions in plant genome. It has been widely used in many plants.But so far, there is no research on the application of FISH in muberry plants.In this research, we first optimized the conditions for preparing the chromosome sectioning of mulberry, and then established the FISH system. We also studied the distribution characteristics of rDNA and telomere sequence on the chromosomes in metaphase of mulberry. The karyotypes of Morus notabilis and Morus yunnanensis were analyzed. The main results are as follows:1. Chromosome numbers and ploidy analysis of Morus L.In the present study, we prepared chromosome samples by means of wall removal and hypotonic treatment. At 8:00-9:00 a.m., when the plant growth is exuberant, the tender leaves were gathered as material. The leaves were pretreated for 3 hours with 8-hydroxy-quinoline at room temperature in dark and then disposed in mixed liquor of cellulose (2.5%):pectinase (2.5%)= 1:1 at 25℃ for 2-3 hours. As results, metaphase chromosomes of twenty-three mulberry species were obtained successfully. We identified the chromosome number of these mulberry germplasm resources and found that most of the mulberry plants had somatic cells with the chromosome number of 28 and there were also mulberries with 14,35,49 and 308 of chromosome number. Mulberry YUN 19 might be mixoploid containing the chromosome number of 35 and 49.2. Establishment of the FISH system in mulberry plantsAfter the slides with metaphase chromosomes were baked in the oven at 37℃ for 2 hours, they were treated with 100 μg/ml RNase A at 37℃ for 1h. These sildes were subsequently incubated in 1μg/ml protease K at 37℃ for 15 min. The optimal amount of hybridization probe was 6 ng/μl.The optimal conditions of denaturation were as follows:1) chromosomal DNA was denatured for 10 min in 70% formamide at 72℃; 2) hybridization solution containing probe was incubated for 6 min at 95℃;3)the slides and were hybridized at 37℃ for about 10 h after they were denatured at 80℃ for 10 min. The results indicated that the number and strength of signal from 5S and 25S rDNA probes hybridized on chromosomes from different mulberry plants were distinct. Based on these data, we found that the number of rDNA signal was not associated with its chromosome ploidy level.The variations of 5S rDNA number were larger than that of 25S rDNA.3.The location analysis of telomeric sequences on chromosomes of Morus notabilisUsing Arabidopsis type telomeres as probes, we localized the telomeric sequences on metaphase chromosome of Morus notabilis by FISH.The results indicated that the majority of chromosomes have telomere signals.Several chromosomes have no telomere signals and some chromosomes have internal telomere sequences.In addition, the length of telomere sequences on each chromosome is different.Our results provide the basic data for the cytogenetic study and karyotype analysis of mulberry plants. They offer a new gist for chromosome evolution, interspecific relationship, and phylogenetic relationship of mulberry, which can guide cross breeding, create new germplasms, and enrich mulberry germplasm resources.