Using spb-CBM9.02_S65 (bz1) as an example

spb = Sorghum propinquum BAC
sbb = Sorghum bicolor BAC
-CBM = cross-hybridizing at high stringency to the Core Bin Marker
9.02 = is the core bin marker for locus 9.2 (as used in the UMC 1998 linkage map from G. Davis et al.)
_S65 = the Short arm cytogenetic locus, 65% of the distance from the centromere (at 0%) to telomere (at 100%); synonyms for this cytological locus include 9S.65 and map position -.65
(bz1) = the maize DNA sequence, bz1 ; used for biochemical or electronic queries of the sorghum genome to select a corresponding sorghum BAC clone (the bz1 locus)

Therefore, the probe named spb-CBM9.02_S65 (bz1) refers to a Sorghum propinquum BAC FISH probe identified as having a high degree of sequence similarity to the maize core bin marker 9.2 and producing cytogenetic FISH signals at 9S.65 on pachytene-stage chromosome spreads.

Probe Nomenclature for Other Maize Markers

Using spb-9.03_L04 (rgpr3235a) as an example

spb = Sorghum propinquum BAC
sbb = Sorghum bicolor BAC
-9.03 = a designation for the maize genetic bin. The FISH probe was selected using a maize marker sequence known to map within the genetic linkage bin 9.03, somewhere between CBM9.03 and CBM9.04. The bins are from Davis et al., 1999.
_L04 = the Long arm cytogenetic locus, 4% of the distance from the centromere (at 0%) to the telomere (at 100%); synonyms for this cytological locus include 9L.04 and map position .04
(rgpr3235a) = the maize DNA sequence, rpgr3235a; used for biochemical or electronic queries of the sorghum genome to select a corresponding sorghum BAC clone

Therefore, the probe named spb-9.03_L04 (rgpr3235a) refers to a Sorghum propinquum BAC FISH probe identified as having a high degree of sequence similarity to the maize marker probe rgpr3235. The name also indicates that the maize marker is located within the linkage bin 9.03, and that the FISH signals were detected at 9L.04 on pachytene-stage chromosome spreads.

Methods (as described by Koumbaris & Bass 2003 and updated by Ferdie Amarillo and Hank Bass)

Sorghum BAC FISH probe selection methods:

Maize marker-selected sorghum BAC FISH probes have been identified in several different ways, depending on ( 1) the method of detection, and ( 2) the probe/query type. Any combination of detection and probe type can be used, as tabulated below.

Sorghum BAC FISH methods:

1) DNA from the sorghum BAC was labeled in situ with digoxigenin or directly with a fluorescent dCTP (Alexa-fluor 546-OBEA-dCTP) by random-primed labeling.

2) Labeled DNA was used as FISH probes on pachytene chromosome spreads from maize chromosome addition lines of oat.

3) FISH was carried out with three-color probe mix containing:

(a) maize total DNA direct-labeled with Alexa488-dUTP to paint the entire maize chromosome.

(b) MCCY, a Cy-5 oligonucleotide probe homologous to the maize CentC repeat sequence, marking the centromere.

(c) Sorghum digoxigenin-labeled BAC hybridization was detected by indirect immmunofluorescence with commercial detection kits in which the FITC reagents were substitued with rhodamine reagents. The Alexa546-labeled probes were detected directly without secondary staining.

4) Images were collected with a digital camera and de-blurred by iterative 3D deconvolution.

5) Chromosomes were straightened and measured computationally.

6) Mean locations are reported as the fractional (relative) distance along a chromosomal arm. Each arm has a length of 1.0 with the centromere being set to 0 for each arm. The length of each arm is divided into 100 centiMcClintocks with, for instance, a cytogenetic position of 9S.33 indicating detection of discrete FISH signals at a point 33% of the distance along the short arm of chromosome 9 from the centromere.

7) Identification of sorghum BACs for use as FISH probes were selected by online searching using the following databases:

(a) http://www.maizegdb.org (for finding an overgo probe used for each marker or locus)

(b) http://chibba.agtec.uga.edu/duplication/bacman/ (BACman utility programs)