Abstract

The resistance gene analogue polymorphism (RGAP) and random amplified polymorphic DNA (RAPD) techniques were used to identify molecular markers linked to yellow strip resistance gene(s) in wheat seedlings. Genomic DNA of five most resistance and five most susceptible F3 families obtained from a cross between Flanders (resistant) and Morocco (susceptible) cultivars were bulked along with DNA from their parents. The genomic DNAs were amplified with RGAP primers designed based on leucine-rich repeat (LRR), nucleotide-binding site (NBS) and kinase domains of a number of resistance genes from database using DNAstar package and random primers for RAPD. The amplified products of both techniques were detected on agarose gel in addition to denaturing polyacrylamide gel for RGAP. Preliminary results indicated that eight RGAP markers and two RAPD markers were present in resistance, but not susceptible, bulked samples. These markers were tested in the parents and RGAP marker P210 and RAPD marker OPF13 showed polymorphism in susceptible and resistant parents. Investigation of the linkage of these markers with individual resistance genes Yr1, Yr3a or Yr4a using near-isogenic lines and linkage analysis in individuals from F2 population is the subject of present investigations.

Introduction

Yellow rust caused by Puccinia striiformis westend f.sp. tritici is considered to be one of the most destructive fungal diseases of wheat in most cool wheat-producing regions. Using resistant cultivars is the best disease control strategy, since it contributes both to reduce environmental contamination and production costs. Historically, race-specific major genes have been used to breed rust resistant wheat cultivars. At present, at least 30 resistance genes have been catalogued (McIntosh et al., 1998), most of them are effective from the seedling stage through the whole life of the plant whereas a few are only effective at the adult stage.

Bringing disease resistance genes and other favorable agronomic traits into a single elite variety by conventional means is very laborious and time-consuming. In the cases which more than one resistance gene is being transferred, it is not achievable because screening for a resistance gene interferes with the ability to screen for others. The lack of virulent isolates for the resistance genes is another frequent problem in disease resistance breeding.

In recent years, DNA-based markers have shown promise in expediting plant breeding procedures. The identification of molecular markers for resistance genes can efficiently facilitate the pyramiding of major genes into a valuable background in less time, making it more cost effective. A number of tightly linked molecular markers for several important disease resistance genes have been identified, e.g. the black root rot resistance gene in tobacco (Bai & Releeder, 1995), the Ml-O (Hinze et al., 1991) and Rh loci (Barua et al., 1993) in barley, Ht1 gene in maize (Bentolila et al., 1991) and Pm3 (Hartl et al., 1993), Lr9 (Schachermayer et al., 1994) and Cre (Williams et al., 1994) genes in wheat.

To identify molecular markers linked to disaese resistance genes, the first step could be evaluation of a number of molecualr markers on genomic DNAs extracted from segregating lines. Examination of the promising marker(s) in the segregating population will be the next step. The aim of this study was identifying molecular markers showing linkage with stripe rust resistance gene(s) in the seedlings of a resistant line using bulked genomic DNA. For this, RGAP and RAPD molecular markers were used in bulked genomic DNAs of F3 plants obtained from a cross between a susceptible and a resistant cultivar.

Materials and Methods

Plant, fungus and inoculation of seedlings
Wheat plants were grown and maintained under controlled conditioned in a growth chamber. The line Bolani was used for proliferation of the stripe rust fungus and 26 standard wheat lines were used to determine the race of fungal isolate used for inoculations. The stripe rust resistant line Flanders (harboring seedling stage resistance genes Yr1, Yr3a, Yr4a and adult plant resistance gene Yr16) and the susceptible line Moroco were crossed. For genetic analysis, 315 plant of the resulting F2 progeny were tested. For marker analysis, a subset of F3 families were used. The plants were artificially inoculated at two-leaf stage with spores of a most aggressive rust isolate from Iran (collected from Moghan). Fourteen and 17 days after inoculation, plants leaves were scored for the infection based on the Mc Neal et al. (1971) scale.

DNA extraction
The method for DNA extraction was adopted form Pitrat (2002). DNA from five of the most susceptible and five of the most resistance F3 families were extracted and combined in equal concentrations to make susceptible and resistant bulks, respectively.

RGAP and RAPD techniques
For the RGAP technique, 24 primers were designed based on conserved sequences (including LRR, NBS and kinase domains) among different disease resistance genes from each class of disease resistance genes. The sequence of the genes were obtained from NCBI internet site and DNAstar software were used for primer design. The primers were used in 19 different combinations with each other and primers designed by Chen et al. (1998). The PCR was performed in conditions described by Chen et al., 1998 and the PCR products were electrophoresed in both agarose and denaturing acrylamide gels and stained with ethidium bromide and silver nitrate, respectively. For the RAPD experiment, 25 random primers were used and the PCR were performed according to Singh et al., 1998. The PCR products were electrophoresed in agarose gels and stained with ethidium bromide.

Results and Discussion

Fungal race and the genetics of the resistance
Using 26 standard lines, the race of the rust isolate was determined as 134E134A+. From 315 F2 plants inoculated, 200 and 44 plants were resistant and susceptible, respectively. According to Chi square analysis, one dominant resistance gene with or without a recessive resistance gene is involved in the resistance of Flanders variety to the rust race 134E134A+.

RGAP and RAPD techniques
Among the 19 different RGAP primer pairs used, 8 pairs (Table 1) produced polymorphic bands between susceptible and resistant bulked DNA samples. Among these primers, the primer pair P2-P10 produced a polymorhic band (600 bp) in resistant, but not susceptible, bulks and parents (Fugure 1a). For confirmation, the experiment was repeated three times and the occurrence of the polymophic band was confirmed. It is suggested that the P2-P10 primer pair may proliferate a part of, or a region close to a resistant gene in the Flanders plant. Genetic analysis on F2 population using the P2-P10 primer pair is required for further confirmation. Also, to determine which resistance gene has possibly been identified, near isogenic lines, each harboring an individual Yr1, Yr3a or Yr4a gene, are to be examined with P2-P10 primers. In RAPD experiment, the random primer OPF13 (5'-GGCTGCAGAA-3') produced a polymorhic band (Figure 1b) in the DNA from susceptible bulked and parent, but not resistant, samples, suggesting its association with a recessive resistance gene in the susceptible line. The experiment repeated several times and similar results was obtained. Using molecular markers, a number of resistance genes have been identified in wheat. Identification of Yr15 and Yr8 using RAPD (Sun et al., 1997; Singh et al., 1998), Yr9 and Yr5 by RGAP (Shi et al., 2001; Chen et al., 2002), YrHs2 and Yrns-B1 by microsatellite (Borner et al., 2000; Peng et al., 1999), YrMoro using STS (Smith et al., 2002) and Yr10 using RFLP (Laroch et al., 1998) are among neumerous attempts in this field.

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