1.12.9
A NEW UNCHARACTERIZED DOMINANT GENE PROTECTS AGAINST THE NL3 STRAIN OF BEAN COMMON MOSAIC NECROSIS POTYVIRUS

RC LARSEN, PN MIKLAS and KD VICTRY

USDA, ARS, Prosser, Washington, USA 99350, USA

Background and objectives
Bean common mosaic disease is caused by two related but distinct potyviruses, bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV), an economically important disease on a global basis. The viruses are transmitted in a non-persistent manner by several aphid species. Because BCMV and BCMNV are often seed-transmitted at very high rates, they are of critical importance in seed-production areas. BCMNV and BCMV exist as a large number of virus strains and are categorized into seven pathogroups based on differential host range response. Six recessive genes and a single dominant I gene are involved in resistance expression to bean common mosaic disease. Unprotected, the I gene conditions a temperature-independent hypersensitive response to BCMNV or an immune/temperature-dependent hypersensitive response to BCMV [1]. Protecting the I gene with one or more recessive genes with resistance to BCMNV or BCMV will prevent, delay or localize the necrosis reaction [2]. Our initial study began using RAPD primer analysis in search of molecular markers for the bc-12 gene. However, during concurrent studies with BCMNV strain NL3 on near-isogenic pinto bean lines (NIL) for the local vein necrosis response, an unusual segregating population was noted. This paper describes this unique population and progress on identification of relevant genetic markers for the gene conditioning vein necrosis.

Materials and methods
A pair of near-isogenic heterogeneous inbred-derived lines were extracted from the pinto bean breeding line P94207. An F2 population was developed from a cross between the NILs. Seedling plants from the F2 and F3 populations were inoculated at the first full primary leaf stage with the NL3 strain of BCMNV representing pathogroup VI. Symptom expression was recorded and a segregation analysis performed. Leaf DNA extracted from samples of three resistant and three susceptible NILs of P94207, respectively, were pooled and analysed using RAPDs. 600 random decamer primers were screened using the bulk-resistant and bulk-susceptible DNA samples in search of a polymorphic segregating marker for the resistance gene to NL3. Selected markers were isolated, cloned and prepared for DNA sequencing.

Results and conclusions
The F3 population yielded the predicted 1:2:1 segregation ratio of true breeding top necrosis, segregating, and true breeding vein necrosis families, respectively. Of the segregating families, a 3:1 segregation ratio of vein necrosis and top necrosis was observed. This ratio indicated that a dominant gene rather than the assumed bc-12 recessive gene was protecting the I gene. RAPD primer analysis generated polymorphic markers G-16 and A-7, estimated at 300 and 1000 bp, respectively. The markers were present in all three individuals of the resistant bulk DNA, and absent in all three individuals comprising the susceptible bulk DNA. The small size of marker G-16 rendered it unreliable under our conditions using RAPD analysis. However, it is being modified for use in SCAR analysis. As additional primers are being screened, marker A-7 is currently being used for analysis of a segregating F2 population. Crosses to test for allelism between the putative new dominant gene and recessive bc-12 gene are being evaluated. In addition, response of the new gene in a recessive I gene background to virus strains representing the seven BCMV and BCMNV pathogroups is in progress. We tentatively propose the symbol for the putative new dominant gene as Bc-v.

References
1. Haley SD, Afanador LK, Miklas PN et al., 1994. Theoretical and Applied Genetics 88, 337-342.
2. Drijfhout E, 1978. Genetic interaction between Phaseolus vulgaris and bean common mosaic virus with implications for strain identification and breeding for resistance. CTA, Wageningen, The Netherlands.