Locating Of An Erianthus Arundinaceus-specific DNA Sequence By In Situ PCR

Karyotype analysis on Erianthus sect. Ripidium YC No.2 (sugarcane parent species) and YC96/46 (sugarcane cultivated variety) were conducted. The karyotype formula of Erianthus sect. Ripidium YC No.2 was 2n=60=60m; the absolute length was 2.26～5.43μm; the relative length was 2.26～5.43; the average arm ratio was 1.19; all the 30 pairs of chromosome were median kinetochores; the karyotype belonged to 1B type. The karyotype analysis of YC96-46 indicated that the karyotype formula was 2n=68=66m (sat) +2sm; the absolute length was 1.83～5.48μm;the relative length wasl.53～5.15; the average arm ratio was 1.38; chromosome No.4 was sub-median kinetochores,others were median kinetochores ; chromosome No.8 had one satellite. The karyotype belonged to 2B type.Both the karyotypes of Erianthus sect. Ripidium YC No.2 and YC96-46 were relatively original in genome evolution.The special STS marker Erf08 of Erianthus was tested to develop a method of direct in situ PCR on chromosomes of Erianthus sect. Ripidium YC No.2,YC96-46 and Badila. The method was developed successfully and its main procedure was as following: The time of cell digested properly with pepsine enzyme before ISPCR was 5 to 10min;The reaction volume of ISPCR was 50μl mix with 200μM dATP,dGTP and dCTP , 72μM dTTP, 4.5mM MgCl2, 0.5mg/ml BSA, 4μM DIG-11-dUTP, 5U Taq DNA Polymerase, 2μM Primer, 1×buffer; The cycling parameters of ISPCR were 10min predenaturalization at 95 ℃, and 25 cycles of 94℃ for 1 min,65℃ for 1 min,72℃ for 2 min,then 10min extension at 72℃;After cycling was completed, the slides were washed in 0. 1 ×PBS for 5 min, and in 0.1×SSC/Tween20 for 2×5min.Several signals were detected on the different phase of the cells of Erianthus sect. Ripidium YC No.2 and YC96-46 used the developed in situ PCR method. The signals on Erianthus sect. Ripidium YC No.2 chromosomes mainly distributed on the telomere. The signals on the chromosomes of YC 96-46 also mainly distributed on the telomere. Combined with the karyotype analysis of YC 96-46, the Erf08 were located on 11...