Use of differential display in conjunction with bulked segregants to target specific genomic loci.
Differential display has proven to be a very successful technique for isolating differentially expressed transcripts. We sought to expand the capabilities of the technique by attempting to isolate cDNAs from specific genomic loci. Two loci of interest to us are the S locus and the MOD locus, both involved in the self-incompatibility phenomenon of Brassica. The S locus is a complex locus for which molecular markers have previously been isolated, and the MOD locus is a single-gene locus for which no markers are available. We used plant material from F2 populations that segregate for two allelic variants of each locus to create two bulks or pools of plants for each differential display screen. Pooling F2 individuals effectively homogenizes background polymorphisms found in the parent plants. RNA was prepared from each bulk and differential display was performed using a kit from GenHunter Corporation (Nashville, TN). One cDNA that segregated completely with the target locus was isolated from each screen. Multiple cDNAs that were linked to each locus were also identified. We have demonstrated that differential display, when used in conjunction with bulked segregants, is a very powerful technique for isolating cDNAs from genomic loci of interest.
Section of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
A L
AL
Casselman
S
S
Ikeda
J B
JB
Nasrallah
M E
ME
Nasrallah
D001937:Brassica / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D003433:Crosses, Genetic; D018076:DNA, Complementary / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D018525:Germination / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics; D006239:Haplotypes / Q000235:genetics; D007473:Ion Channels / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics
D018076:DNA, Complementary; D005819:Genetic Markers; D006023:Glycoproteins; D007473:Ion Channels; C106143:MOD protein, Brassica rapa; D010940:Plant Proteins; C064156:self-incompatibility locus specific glycoprotein, Brassica; D011494:Protein Kinases; C079832:S-receptor kinase