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A Cleaved Amplified Polymorphic Sequence (CAPS) Marker Associated with Root-Knot Nematode Resistance in Sugarbeet

^a USDA-ARS, Red River Valley Agricultural Research Center, Fargo, ND 58105
^b USDA-ARS, Crop Improvement and Protection Research Laboratory, Salinas, CA 93905

View larger version (82K): [in a new window]   Fig. 1. Generation of a DNA marker associated with sugar beet resistance to root-knot nematode. Bulked DNA samples prepared from susceptible (S1, S2, S3) and resistant (R1, R2, R3) sugar beet plants were subjected to RAPD analysis using the decamer primer pair OPA02 and OPB01. Amplified DNA products were separated on a 1.6% (w/v) agarose gel, stained with EtBr and photographed. The approximately 0.6-kb band seen only in the lanes representing the resistant bulk samples and indicated by the arrow (NEM06) was cloned for sequence analysis and subsequent conversion to a CAPS marker. The 100-bp ladder was included as a size standard (lane M) with the sizes shown listed in kilobasepairs.

View larger version (49K): [in a new window]   Fig. 2. Sequence analysis of the 0.6-kb amplified DNA marker associated with resistance to root knot nematode. In A, the complete nucleotide sequence is shown with the decamer primer binding sites (underlined) and the subsequent CAPS marker primer binding sites (block bold text) indicated within. The four nucleotide sequence (bold italics) comprising an MseI resistriction site polymorphism difference between alleles of the marker is also shown. Translation of a portion of the DNA sequence into encoded protein sequence (B) indicating similarity to the proteins encoded by the PERIANTHA gene from Arabidopsis thaliana (A.t.) and the NIP1 gene from Lycopersicum esculentum (L.e.), both putative transcription factors. An alignment (C) of a portion of the NEM06 marker sequence amplified by the Nem06fwd and Nem06rev primers from both susceptible and resistant sugar beet. The four-base recognition sequence for the restriction enzyme MseI is shown in bold with boxes surrounding the nucleotide difference between the alleles that determines the inability ("G") or ability ("A") for this enzyme to cleave the DNA.

View larger version (51K): [in a new window]   Fig. 3. Marker status of sugar beet plants rated for resistance or susceptibility to root-knot nematode. Amplified Nem06 marker DNA from 16 sugar beet plants either susceptible or resistant to root-knot nematode was digested with MseI and the products were separated by electrophoresis on a 5% (w/v) polyacrylamide gel. Arrows denote the migration of the marker allele (r) that is coupled with resistance and the marker allele (s) associated in repulsion with resistance. The 100-bp ladder was included as a size standard (lane M) with the sizes shown listed in kilobasepairs.