Development and characterization of SCAR markers linked to the citrus tristeza virus resistance gene from Poncirus trifoliata.

Twelve new dominant randomly amplified polymorphic DNA (RAPD) fragments associated with a single dominant gene for resistance to citrus tristeza virus (CTV) were identified using bulked segregant analysis of an intergeneric backcross family. These and eight previously reported RAPDs were mapped in the resistance gene (Ctv) region; the resulting localized linkage map spans about 32 cM, with nine close flanking markers within 2.5 cM of Ctv. Seven of 20 RAPD fragments linked with the resistance gene were cloned and sequenced, and their sequences were used to design longer primers to develop sequence characterized amplified region (SCAR) markers that can be utilized reliably in marker-assisted selection, high-resolution mapping, and map-based cloning of the resistance gene. All seven cloned RAPDs were converted successfully into SCARs by redesigning primers, optimizing PCR parameters (especially the annealing temperature), or digesting amplification products with restriction enzymes. Four of the seven remained dominant markers, displaying presence-absence polymorphism patterns; the other three detected restriction site changes or length variations and thus were transformed into codominant markers. Two genomic regions rich in variability were also detected by two codominant SCAR markers.

[1]  M. Roose,et al.  Origin and Inheritance of Dwarfing by the Citrus Rootstock Poncirus trifoliata `Flying Dragon' , 1995 .

[2]  P. Stam,et al.  Construction of integrated genetic linkage maps by means of a new computer package: JOINMAP. , 1993 .

[3]  W. Rychlik,et al.  A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. , 1989, Nucleic acids research.

[4]  D. D. Kosambi.  The estimation of map distances from recombination values. , 1943 .

[5]  J. Welsh,et al.  Fingerprinting genomes using PCR with arbitrary primers. , 1990, Nucleic acids research.

[6]  G. Martin,et al.  Map-based cloning of a protein kinase gene conferring disease resistance in tomato. , 1993, Science.

[7]  M. Daly,et al.  MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. , 1987, Genomics.

[8]  F. Gmitter,et al.  363 INHERITANCE OF CITRUS NEMATODE RESISTANCE AND ITS LINKAGE WITH RAPD MARKERS IN CITRUS , 1994 .

[9]  R. Michelmore,et al.  Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[10]  W. Rychlik,et al.  Selection of primers for polymerase chain reaction , 1995, Molecular biotechnology.

[11]  K. Livak,et al.  DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. , 1990, Nucleic acids research.