Use of Molecular Marker for Assay Gene Dosage Resistant Gene to Rhizomania Disease (Rz1) in Sugar beet (Beta vulgaris L.)
Nouhi Ali Askar,
Hagh Nazari Ali,
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 dont effecting additional one
allele (R) to resistance again Rhizomania disease.
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
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.
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 didnt
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 didnt 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).
||Mean of absorbance (ELISA) for Rz1Rz1 and Rz1rz1 genotypes
with use from Op-AN9600r marker in F2-A1-110 population
|n1: No. of homozygous genotype, n2: No. of heterozygous
genotype, NS: Non-Significant
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.
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