Research Areas

Plant Breeding and Biotecnology 2004-2007

Researcher: Pedro Antonio Arraes Pereira

Soybean Rust

Soybean rust is caused by two different fungal species. One is Phakopsora pachyrhizi, the causal agent of the Asian soybean rust. This pathogen is very aggressive and can cause severe damage and significant yield losses on soybeans. The other species is P. meibomiae that usually causes mild symptoms on soybeans. P. meibomiae is found only in the Americas.
Unlike other rust pathogens, P. Pachyrhizi and P. meibomiae infect a very broad range of plant species, including common beans (Phaseolus vulgaris). It has been reported that this plant species is a natural host to both of these pathogens. It is known that some common bean cultivars, such as red kidney beans, are very susceptible to P. meibomiae. Similarly, some dry bean cultivars appear to be very susceptible to P. pachyrhizi, at least under greenhouse conditions. There is no published data showing the reaction of common bean cultivars to P. Pachyrhizi under field conditions and whether this pathogen could be an economic threat to common bean production in general or to only some specific cultivars. The purpose of this research is to determine the reaction of some important bean cultivars grown in Brazil and the United States to P. pachyrhizi, the Asian rust pathogen, under field conditions in Brazil and also South Africa. The results from South Africa and Brazil were compared here with unpublished results obtained previously under greenhouse conditions in the US . The soybeans planted in Brazil developed severe ASR symptoms. In Goiania, the soybeans had 70 % average severity while in Rio Verde the average severity was 60 %. In both locations, the common bean cultivars CNC, PI 181996 and Aurora, had no visible ASR symptoms, while the other cultivars had very mild symptoms.


Identification of SSR markers linked to rust resistance genes in common beans

Molecular markers linked to genes that confer disease resistance are very useful in marker-assisted selection (MAS) and for pyramiding or combining various disease resistant genes in a bean cultivar. The purpose of this project is to find molecular markers linked to the gene in the Andean bean PI 260418 that is resistant to all but one of the 90 races of the bean rust pathogen identified and maintained at Beltsville. Bean rust caused by Uromyces appendiculatus is one of the most important diseases of common bean (Phaseolus vulgaris L.) in the world and U. appendiculatus is a highly variable plant pathogen. Understanding this variability is a prerequisite for identifying resistance genes and deploying them into more stable cultivars to different regions. Differential host genotypes are commonly used to distinguish races of rust pathogens. A set of 12 standard bean rust differentials lines or cultivars has been used to distinguished races and pathotypes of U. appendiculatus .Middle American germplasm offers the broadest spectrum of resistance genes, although the reaction of Middle American germplasm to population of U. appendiculatus is more variable than that Andean (Araya, C. M. 1996; Sandlin, et al. 1999).
The microsatellite are polymerase chain reaction (PCR) based on markers that have been developed for a wide range of plant species including many commercial crops. The utility of microsatellites derives from the fact that they detect length polymorphisms at genetic loci that have simple sequence repeats (SSR’s). The objective of the present work is to find SSR markers that are linked to the major rust resistant genes.
The study was conducted using the beans lines Pinto 114 and PI 260418. Several primers were evaluated to identity primers that were polymorphic between the two bean parents. 602 bean SSR primers were evaluated and 158 primers were identified as polymorphic for these two beans lines. SSR’s generated for soybean were also tested from 715 soybean SSR’s only 90 presented products in the gels but none of them identified polymorphisms between these bean lines (Table 8) The bean SSR primers derived from genomic sequences have greater number of primers that generated products when compared with SSR’ derived from EST sequences. Among the genomic SSR’s primers 71.9% generated products compared with 59.2 % of EST primers that generated products. Furthermore 27.7% of the genomic primers detected polymorphism between the two parents compared with 21 % of the EST primers. In the case of the SSR primers published in the literature 91.1% of them generated product and indeed 29.5 % of them identified polymorphism between the bean parents.
Two SSR’s were found to be closely linked with the region that confers resistance to all rust isolates present on the PI 60418. Although the two rust resistance genes were linked together 20cM apart.
In order to identify markers linked to the other important resistant genes to bean rust. The following populations were advanced to F2 generation to score for each specific rust race and collect leaves from the F2 individual plant to allow the evaluation of the segregating population for the different SSR’s primers.


SNP DISCOVERY IN P. VULGARIS FROM SOYBEAN-DERIVED PCR PRIMERS SETS

Single nucleotide polymorphisms (SNP’s) represent the most common variations across a genome [1], they occur at a frequency of about one SNP in 1000 nucleotides in genomic DNA [2] and they can be used to directly detect alleles responsible for a trait of interest. SNP’s have several uses in genetics including the detection of alleles associated with heritable human diseases [3] and inferences of population history [4]. SNPs can also be used for fingerprinting and the generation of genetic maps, In contrast, every SNP in context with its surrounding genomic sequence is unique. SNPs can mark functionally important allelic differences, and SNPs that flag individual alleles of known genes have been used widely as molecular markers.
Recent work at Soybean Genomic Laboratory ARS/USDA, Beltsville under the leadership of Dr Perry Cregan reported the results of SNP discovery in P. vulgaris via the analysis of 1204 soybean-derived PCR primers sets. A total of 188 primer sets amplified a single PCR product in common bean and 97 of these contained a SNP as determined by the DNA sequence analysis of the P. vulgaris genotypes, BAT93, Jalo EEP558, DOR364, and G19833 (Dr. Perry Creagan`s information). Approximately 9,500 soybean-derived primers sets are available at the USDA, Beltsville. Analysis of these in P. vulgaris should translate into the development of useful SNP markers in common bean. Dr. Glaucia S. Cortopassi Buso, researcher of Embrapa Genetic Resources and Biotechnology began her work at the Soybean Genomic Laboratory at USDA-Beltsville to use these soybean-derived primer sets to discover additional SNPs in P. vulgaris. She tested the amplification of 864 (1728 considering forward and reverse) SNP soybean primers, at annealing temperatures of 480C and 580C, in P. vulgaris (Jalo EEP558). The percentage of amplification of a single band in P. vulgaris was in average 46.28%, where 311 primers amplified at 48 and/or 580C and 93 amplified at 580C. Those primers were used to amplify 4 genotypes (BAT93, Jalo EEP558, DOR364, and G19833) and the amplification product was sent to sequencing, giving a total of 3224 sequences. These sequences were analyzed to certify the existence of SNPs, and were produced by 403 primers, forward and reverse, in 4 genotypes. From the 403 analyzed fragments 91 had at least one SNP (22.58%). The fragments had 1 to 23 SNPs, producing a total of 304 polymorphisms of single base for beans.