Optimization Of Chromosome Sectioning And Establishment Of FISH And GISH Of Lagerstroemia L.

Crape myrtle (Lagerstroemia indica L.) is an important ornamental tree species of Lythraceae. The investigation on Lagerstroemia mainly focused on germplasm resources, propagation, cultivation and landscape application. However, there was little research on cytology, because the chromosomes are very small and the cytoplasm is very thick. There were only simple reports of chromosome morphology and karyotype. There was no research on the structure of genome and molecular cytogenetic. Fluorescence in situ hybridization (FISH) is based on the principle of complementarity, which can be used in obtaining the precise position of modified nucleotide numerator probe on chromosomes, and could offer recognized stamps for small chromosomes. In this paper, we first optimized the chromosome sectioning of Lagerstroemia, and then established the FISH and GISH system. The results were as follows:1. Optimization of chromosome sectioning of Lagerstroemia L:new stem tip fixed in modified Carnoy solution (EtOH:HAC:THMs=5:3:2) at-20℃overnight performed best in sectioning. The best treatment of conventional squash method is disentangling2h in mixed liquor of concentrated hydrochloric acid and EtOH (1:1). The best treatment of enzyme hydrolysis method is disposing4-5h in mixed liquor of cellulose (2.5%):pectinase (2.5%):Protease K (0.01%)=2:1:3. The conventional squash method was appropriate for chromosome counting and observation.2. Establishment of FISH system of Lagerstroemia:Chromosome pretreatment:drying2h in65℃oven. Incubation1h with0.1mg/ml RNA at37℃. Incubation10min with0.1mg/ml pepsin at37℃.The hybridization mixture consisted of3μl probe in20μm system. The mixture was denatured for10min in metal bath at95℃and chilled on ice for10min. Chromosomal DNA on the slide was denatured at85℃for3.5min. Signal detection:using20μl2mg/ml DAPI dyeing5min. The results indicated that the45SrDNA loci on Lagerstroemia were at the end of one chromosomal short arm. The loci was found on the second chromosome of Lagerstroemia indica, first chromosome of Lagerstroemia limii, the fourth chromosome of Lagerstroemia caudata and the second chromosome of Lagerstroemia fauriei. We also obtained the karyotypes of the four Lagerstroemia species.3. GISH system of Lagerstroemia was established and it was found that there were hybridization signals both of L. indica and L. caudata on the chromosomes of F1.Which proved that these plants were real hybrids.The results laid the foundation for molecular cytology studies on Lagerstroemia.