Use of Markers as a Tool to Investigate the Presence of Disease Resistance Genes in Common Bean Cultivars

The molecular marker-assisted selection has a great potential as a breeding tool in common beans. The identification of RAPD markers allows association (pyramiding) of genes for disease resistance or the identification of high potential cultivars with different resistance genes that were not previously identified due to limitations in the screening methodology. In this study previously identified molecular markers were used as a tool to investigate the presence of disease resistant genes as well as to identify pyramided genes that are present in the lines tested in the elite lines trials coordinated by Embrapa Rice and Beans. Fourteen out of 21 elite lines possessed different molecular markers linked to disease resistant genes for anthracnose, angular leaf spot, rust and bean golden mosaic virus. Lines FEB 163 (5) and TB 94-01 (8) presented seven molecular markers linked to different resistance genes. It was not possible to detect the presence of any known sources of disease resistance genes, based in the pedigree of lines LM 93204319 (12), LM 93204328 (13), LM 93203246 (18) and PR 93201472 (21).

The identification of molecular markers linked to resistance genes allows an indirect selection, because the expression of the marker is not masked by epistatic interactions (Kelly, 1995).The identification of a number of RAPD markers allows pyramiding genes for disease resistance, or the identification of potential lines with different resistance genes which could not be previously identified due to difficulties in accomplishing multiple inoculation tests with different pathogens in just one individual.RAPD markers can facilitate the identification of potential lines with pyramided resistance genes especially if they are tightly linked to the locus containing the resistance allele.
The objective of this study was to use previously identified RAPD markers as a tool to investigate the presence of disease resistance genes, as well as to identify pyramided genes from resistance sources to diseases such as anthracnose, angular leaf spot, rust and bean golden mosaic virus, that are present in the pedigree of lines tested in the bean regional elite trials coordinated by Embrapa Rice and Beans.

MATERIAL AND METHODS
The present study included 21 elite bean lines from the regional trials coordinated by Embrapa Rice and Beans (Table 1).Fifteen days after planting, plant material from each line was collected, identified and stored at -80 o C. DNA extraction was conducted according to Doyle and Doyle (1990) and DNA amplification reactions were achieved according to Williams et al. (1990).Amplification cycles, product analyses and band visualization were similar to those mentioned by Alzate-Marin et al. (1999b).
To investigate linkage relationships between resistance and molecular markers, the pedigree of all genotypes involved in the development of each elite line was compared (Table 1).Based on this information and through a literature review, a second list of all RAPD molecular markers linked to known resistant genes in each genotype was developed (Table 2).As a result, RAPD molecular markers linked to resistance genes sources present in the 21 elite lines tested in the Bean Regional Trials were validated according to the information showed in Table 2.

RESULTS AND DISCUSSION
A summary of the results obtained in this study is shown in Table 3.In general, previously identified molecular markers were efficient in validating the presence of resistance genes from Cornell 49-242, Honduras 35 (Ouro Negro), Tara, Jules, NEP 2, Pompadour Checa and Garrapato.Marker OPF10912C linked to a resistance gene in the cultivar Tlalnepantla 64 (=PI 207.262) was less specific due to its distance (11.5 cM) from the gene (Castanheira et al., 1999) (Table 3).Different markers, in repulsion (Young and Kelly, 1997) or in coupling phase (Haley et al., 1993;Castanheira et al., 1999), linked to anthracnose and rust resistance genes originated from the same primer OPF10 have already been identified.
Amplifications with the molecular marker OPH20450C (Adam-Blondom et al., 1994), happened to be more efficient in tagging the gene Co-2 from the cultivar Cornell 49-242 than the marker OPQ041440C (Young and Kelly, 1996a).As mentioned by Young and Kelly (1996a) Amplifications with the co-dominant marker OPR02 570C/530R , linked in repulsion phase (band of 530 bp) to the recessive bgm-1 gene of BGMV from the resistance source Garrapato, showed that the two bands, linked in coupling and repulsion phase were observed in line FEB 163 (5).The presence of both alleles indicates that the recessive gene locus is heterozygous and susceptible in FEB 163 (+/+=Bgm-1 bgm-1).However, there must be other genes that provide susceptibility or resistance to the bmg-1 originated from the parents of the line FEB 163.According to Urrea et al. (1996), cultivar Porrillo Sintetico exhibits a phenotype +/-(homozygous susceptible: Bgm-1Bgm-1) and the cultivar Garrapato -/+ (homozygous resistance: bgm-1bgm-1) for bgm-1 gene, when amplified with the marker OPR02 570C/530R .FEB 163 is derived from these two parents, consequently the two markers generated by the primer OPR02 are probably indicating the presence of different genes in FEB 163 (Table 1).
DNA amplified from lines LR 9115398 (1), AN 9021334 (9), and AN 9021336 (10) with the SCAROPF10 1050C marker (linked to anthracnose and rust resistance genes), SCAROPBA08 530C and OPX11 530C (linked only to rust resistance genes) (Corrêa, 1999;Vinhadelli et al., 1997) 8) possesses the marker linked to the gene Ur-3 (Table 3).This observation shows a high specificity of this marker for the Ur-3 gene and the absence of alleles of this gene among the other two cultivars.
DNA from lines RAO 33 ( 6) and LR 93201684 (20) that have the cultivar Pompadour Checa as a parent and from line PR 93201472 (21) (Pompadour/Irai), was amplified with primer OPJ13, which tags the rust resistance gene Ur-9.Figure 1 shows that the 1800 bp marker linked to Ur-9 in the cultivar Pompadour Checa is present in lines RAO 33 and LR 93201684 but absent in line PR 93201472.Although cultivar Pompadour Checa was selected from Pompadour (Voysest, 1983;2000), our results show that there must be some differences between these cultivars and PR 93201472.Field data show that lines RAO 33 and LR 93201684 were resistant to rust while cultivar PR 93201472 was susceptible.Lines RAO 33 and LR 93201684 possess the marker OPJ13 1800C while the line PR 93201472 does not (Figure 1).The absence of the marker OPJ13 1800C linked to Ur-9 gene and the susceptibility to rust under field conditions indicate that Pompadour, used as parent of PR93201472, is unlikely to have been the parent of Pompadour Checa.(Young and Kelly, 1996a), OPH20 450C (Adam-Blondon et al., 1994) Angular leaf spot: SCARN02 890C (Nietsche et al., 2000) I and bc-u genes 3/ Middle American (Vasconcelos, 1996).Anthracnose and angular spot resistance source and differential cultivar.
Our work showed a practical application of molecular markers as a tool in the selection process directed toward the commercial release of new cultivars.Our study also indicated that pedigree information has a fundamental importance in the process of validating previously identified disease resistance gene sources.1).Arrow indicates the 1800 bp band linked in the coupling phase to the rust resistance gene Ur-9 present in cultivar Pompadour Checa.

Figure 1 -
Figure1-Electrophoretic analysis of amplification products obtained with primer OPJ13.The first lane correspond to lambda DNA cut with EcoRI, BamHI, and HindIII (m.w.marker).Lanes 6, 20 and 21 correspond to the amplification products of elite lines from the Regional Trials coordinated by EMBRAPA Rice and Beans (Table1).Arrow indicates the 1800 bp band linked in the coupling phase to the rust resistance gene Ur-9 present in cultivar Pompadour Checa.

Table 1 -
Seed color and pedigree of the 21 elite lines of the Bean Regional Trials coordinated by Embrapa Rice and Beans.

Table 2 -
General information on previously identified RAPD markers present in cultivars/lines involved in the breeding program to develop the elite lines of the Bean Regional Trials coordinated by Embrapa Rice and Beans.

Table 3 -
Summary of DNA amplification of the Bean Regional Trials elite lines coordinated by EMBRAPA Rice and Beans using previously identified RAPD markers linked to resistance genes.