Use of Molecular Marker for Assay Gene Dosage Resistant Gene to Rhizomania Disease (Rz₁) in Sugar beet (Beta vulgaris L.)

This study verified the effect of infected soil on resistant plants varieties to rhizomania disease and with using a molecular marker discriminated homozygote from heterozygote genotypes which this theory performed with use of infected soil that was provided from infected fields then greenhouse test was done to identify resistant and susceptible plants. Extracted DNA from leaves of resistant and susceptible plants was bulked to provide two bulks for resistant and susceptible plants then verified affect of allele (R) on increase resistance to Rhizomania disease. The F₂ population for study obtained from cross between Holly1-4 as resistant parent and annual cultivar as susceptible parent. In this study three-hundred RAPD primers was used for detection of molecular markers linked to resistance gene. Finally the gene (Rz₁) was tagged with using of two RAPD primers and one of the markers was OP-AN₉ which was mapped 13.7 cM apart from Rz₁ gene in repulsion phase. Then with t-test statistical method and repulsion marker verified equal of variations related to two population heterozygote and dominant homozygote genotypes. Finally accepted hypothesis equal of variation between two population at p≤0.05. This result show don’t effecting additional one allele (R) to resistance again Rhizomania disease.

INTRODUCTION

The Rhizomania disease is the important disease sugar beet in world and is reported from USA, European countries and many countries of Asian including Iran (Lennefors et al., 2000; Wisler et al., 1997; Nielsen and Nicolaisen , 2001). This disease affect on decrease yield sugar in field (Wisler et al., 1997; Johnson et al., 1995). Rhizomania refers to the excessive hairy root proliferation that results from infection by the Necrotic yellow vein virus (BNYVV, genus Benyvirus). This virus transmitted by the soil-borne plasmodiophoride-like fungus, Polymyxa betae Keskin (Tamada et al., 1990; Keskin, 1964). Source of resistance to rhizomania were found in Holly sugar beet company source (Lewellen et al., 1987). Resistance in Holly is simply inherited by a single dominant gene (Rz₁) (Lewellen et al., 1987; Scholten et al., 1996, 1997; Barzen et al., 1997; Readfearn and Asher, 1997; Asher and Kerr, 1996). Also resistance to BNYVV has been obtained in several Wild Beet (WB) accession B. vulgaris subsp. maritima originally collected from France, UK, Denmark and Italy (Whitney, 1989; Lewellen, 1995). For example WB42 is thought to have been originally collected in Denmark by Viggo Lund in the 1950 (Lewellen, 1995). Resistance in WB42 is inheritance by another dominant gene (Rz₂) (Lewellen, 1995; Scholten et al., 1997; Francis et al., 1998; Francis, 1999). In this study, we verified the effect of infected soil on resistant plants varieties to rhizomania which this theory performed with use of infected soil that was provided from infected fields.

MATERIALS AND METHODS

Studies on the inheritance of resistance to BNYVV were performed in greenhouse of Sugar Beet Seed Institute (SBSI) in Karaj, Iran (2004) with the resistant sugar beet accession B. vulgaris subsp. vulgaris Holly1-4, which is a selection from the Holly source (Lewellen et al., 1987) and the resistant wild beet accession B. vulgaris subsp. maritima WB42. Both accessions are diploid with 2n = 18. Plants of the resistant wild beet accession WB42 also were crossed in pairs with susceptible sugar beet germplasm 261 and annual beet accession. Resistant F₁ plants obtained after crosses of Holly1-4 with the susceptible sugar beet germplasm 261 and annual beet accession were selfed to product F₂ seeds.

Greenhouse Test and ELISA Test
A greenhouse test for screening sugar beet for resistance gene to BNYVV ( Paul et al., 1992) was used in the present study. Seed sowed in autoclaved (121°C, 1.5 h) sand. Seedlings were transplanted at 4 leaf stage to uniform mixture of infested soil was collected from Shiraz, Iran that contained the A type of BNYVV. Regina germplasm was used in all of the tests as negative and positive control. To produce negative control, seedlings of Beta vulgaris sugar beet cultivar Regina were transplanted into sand (not artificially infected plants as negative controls).

To produce positive controls, seedlings of Regina were transplanted into an uniform mixture of infested soil and sand (3:7 V/V). Infected plants as positive controls. Plants were watered twice a week with 30 mL of 0.2 diluted (Hoagland and Arnon, 1950) solution (pH 7.0). Inoculation experiments were performed in the greenhouse at 25/17°C (day/night). Rootlets were analysised for the virus by standard Double Antibody Sandwich ELISA (DAS-ELISA) as was described by Clark andAdams (1977). This experiment was used a commercial polyclonal antiserum and BNYVV infected N. clevelandii leaf (Bioreba AG, Switzerland). The 60 internal wells of micro titer plate (polystyrene Nunce. Flat bottom 442404) were used for ELISA, while the outer wells were filled with PBS-Tween 20 during all steps of the ELISA procedure. All samples were read by Lab system Multiskan EX 355 at 405 nm. Plants were considered susceptible if their samples would show an absorption value more than two time value of the negative control.

DNA Extraction
Genomic DNA was extracted from frozen leaves from individual plants of F₂-A1-110 and F₂-93 populations following the procedure of Saghai-Maroof et al. (1984). DNA concentrations were estimated by spectrophotometery, after that PCR was performed in a total volume of 25 μL containing, 50 ng genomic DNA, 0.2 mM each of dATP, dCTP, dGTP, TTP, 25 ng primer (Operon kit), 2.5 μL 10x Reaction buffer (100 mMTris-HCl, pH = 9; 500 mM KCl), 1 unit Taq Polymerase (Smar Taq). DNA amplification was performed in a thermocycler (Biometra T3) in PCR reaction tubs. The thermal cycles used were: 1 cycle of 5 min at 94°C, followed by 40 cycle of 45 sec at 94°C, 45 sec at specific temperature and 80 sec at 72°C, then finally 1 cycle of 10 min at 72°C for final extension. Amplified products were separated by gel electrophoresis using 1.2% agarose gel with TAE buffer and staining with ethidium bromide.

Bulk Segregant Analysis (BSA)
BSA technique (Michelmore et al., 1991) was performed on bulks of DNA of 10 of the most resistant (with virus concentration maximum 0.3 ng mL^-1 virus) and of the most susceptible plants (with virus concentration at least 0.6 ng mL^-1 virus) for any population. Primers which had amplified a DNA fragment in only one of the bulks were confirmed on the same set of bulk, followed by PCR on ten individual resistant and ten susceptible plants. RAPD markers with the best linkage to resistance gene were evaluated further on additional number of individual plants.

RESULT AND DISCUSSION

To identify RAPD marker linked to genes for resistance to BNYVV (Merdinoglu et al., 1995), DNA bulks were found to be mixture of the most resistant and most susceptible plants of the segregating families of Holly1-4 and WB42 for each set of collection 300 Operon primers were screened. Between 10-20 primers amplified RAPD markers both in a resistant and a susceptible bulk. These primers were examined further on individual plants. The primer Op-AN₉ had generated DNA fragment that was found to be linked to the susceptible locus in F₂-A1-110 population and primer Op-X₉ that had generated DNA fragment 1150 base pair, but no primers found for F₂-93. For this reason, we stopped investigation on F₂-93 population and continued this study on F₂-A1-110 population and primer Op-AN9 was examined on individual plants.

The marker Op-AN9 amplified 600 bp fragment that was linked to Rz₁ gene. This marker was mapped for Rz₁ gene in 13.7 cM (centi Morgan) apart from Rz₁ gene in repulsion phase by Mapmaker Software version 3.0. In study of Barzen et al. (1997) and Scholten et al. (1996) this distance was less of this results because method which they used for estimation of distance was relative of plants that didn’t have 600 bp band on gel electrophoresis to total plants while in our study distance was estimated as relative of two-time of plants that didn’t have 600 bp band on gel electrophoresis to total of plants. In fact we considered heterozygote genotypes in our estimation for more precision. Also plants that were used by Scholten et al. (1996) were 60 individual plants while in our study they were 160 individual plants. This results was supported by Amiri et al. (2003). Also soil that we used concentration of virus was more than soil that was used by Scholten et al. (1996) and Amiri et al. (2003), thus we should have increased threshold of resistance between susceptible and resistance plants that this threshold is less in study of Scholten et al. (1996) and Amiri et al. (2003).

Study Effect of Gene Dosage Related to (R) Allele
With using of information related to this marker and results of ELISA test, it was possible to discriminate between dominant homozygous and heterozygous genotypes. Accordingly, this marker was evaluated on 30 individual of resistant plants of F₂-A1-110 population and discriminated dominant homozygous and heterozygous plants. Then different absorbance means of ELISA test related to both dominant homozygous and heterozygous plants were evaluated with T-test statistical method by computer programming MSTATC (Table 1). First hypothesis was equality absorbance means both dominant homozygous and heterozygous plants. This hypothesis at p≤0.0 5 was accepted. As regard to results, we concluded that the presence of a resistant allele has not effected on resistance increase to disease and both Rz₁Rz₁ and Rz₁rz₁ genotypes were equal in resistance. Also this result demonstrated the dominant character of the resistance. Present results were supported by Scholten et al. (1996) and Amiri et al. (2003).

ACKNOWLEDGMENT

This study was supported by Sugar Beet Seed Institute (SBSI) and university of Zanjan, we are thankful for the collaboration in provide greenhouse and laboratory research activity Dr. Nouroozi and Dr. Mahmoodi (Assistant-Professor) and other memberships of SBSI.