The sugar beet pathogen Beet Mild Yellows Virus was inoculated using the aphid Myzus persicae into the model plant, Arabidopsis thaliana. This led to two plants which expressed possible resistance in the form of BMYV-ss-Sna-1 and susceptibility BMYV-SS-Col-0. After crossing Col-0 and Sna-1 to create an F1 this was self fertilised to create an F2 generation which exhibited a 3:1 ratio of susceptibility to resistance. The susceptibility locus, was investigated by using the technique simple sequence repeat (SSR) polymorphism. Mapping of the resistance locus was first attempted by randomly selected SSR markers covering chromosomes 1 to 5. It was subsequently shown that the resistance gene resided on chromosome 4. Linkage was demonstrated with the microsatellite markers CIW6 and G4539 on chromosome 4 which were mapped relative to the resistance gene BMYV-ss. This discovery proves significant as further markers can now be used to flank this region and further analyse the resistance gene in Arabidopsis thaliana. (This abstract is a bit confused in places- more care needed to use the correct terminology).
INTRODUCTION:
Sugar beet is a crop of prime importance due to its sugar properties which today are produced for food, animal fodder, and more recently bio-fuel. The British sugar beet industry began during the early 1900's, however, the first extraction of sugar began in 1747, by the Prussian chemist Andreas Sigismund Marggraf, who used alcohol to extract sugar from beets. Nonetheless this method did not enter industrial scale production until after the blockade of continental ports occurred during the Napoleonic wars, which consequently prevented export supplies of sugar cane from the West Indies. This encouraged Franz Karl Achard to begin selective breeding from White Silesian beet, which was then a fodder crop. Eventually the first sugar beet factory was formed in Prussia in 1801, which contained at the time only about 4 per cent sugar incorporated into the beet.
In the UK the beet industry started during the 1900's with the first factory being built by the Dutch at Cantley in Norfolk in 1912. This then escalated to 17 factories during the 1920's and the crops were processed by 13 autonomous companies. Then during 1936 those factories were amalgamated by the Sugar Industry Act to form one big multinational corporation, British Sugar PLC. They were to manage the entire domestic crop; a development which marked a significant stage in the progress of the UK beet sugar industry.
Today British Sugar are contracted with 4,000 farmers and between September through until March as much as seven and a half million tonnes of sugar beet are delivered to the factories. In one factory alone 14,000 tonnes of sugar beet are processed in one day.
Fig 1. Shows the sugar processing and industrial use of Sugar Beet at the British Sugar factories.
British Sugar produces over 1 million tonnes of sugar in the UK and an additional 450,000 tonnes of animal feed from sugar beet pulp. Furthermore they provide other vital aspects through the production of sugar beet such as recycled stones for building; lime for soil conditioning and soil for landscaping. They also use combined heat & power plants to export enough electricity for 350,000 people and use the combustion gases to grow 80 million tomatoes. More recently they helped to maintain fuels by investigating alternative substitutes for inevitable depletion of fossil fuels. British Sugar has invested in the UK's first bio-ethanol plant, producing 70 million litres of renewable fuel through the production of Sugar Beet, to help prevent a global economical crisis.
It is clear then that sugar beet production will always be a necessity; however it is not a simple crop to grow as it suffers from many constraints. For the duration of its first growing season it is used for commercial harvesting and can produce as large as 2kg tubules which contain 15-20% sucrose weight. During its second season the nutrients in the roots are used to generate flowers and seeds however this must take place in various locations as their frost resistance is poor but the plants need to have colder weather to flower and produce seed. For the period of its seedling stage it is a poor competitor with weeds and can also be damaged by Heterodera schactii.
Sugar beet also suffers from viral infections which inhibits growth and sucrose production.
Beet Mild Yellows Virus (BMYV)
Sugar beet crops worldwide are affected by several different aphid-transmitted yellowing viruses. In Europe the main threat is through viral infection of the closterovirus Beet Yellows virus and the polerovirus BMYV(Beet Mild Yellows Virus). BMYV is found frequently in northern and western parts of Europe whereas BYV (Beet Yellows Virus) is more commonly found in southern Europe (Smith, 1987). It affects beet leaves which yellow prematurely and causes a reduction in photosynthetic activity as well as decreasing root weight and sugar concentration in infected plants (Smith and Hallsworth 1990). BYV has a large affect on yield production if the virus infects the plant during early growth, decreasing the overall yield by 30 percent. This has been estimated to cost the national sugar industry 24,700 t/year which is the equivalent to 1.8% of the countries yield and a total of £5.5 million.
Fig 2 shows characteristic yellowing of the leaf. In all cases, BMYV is restricted to the vascular tissue in stems and petioles of N. benthamiana. This is consistent with the observation that BMYV replication and movement are limited to the vascular tissue.
BMYV is a member of the genus polerovirus which is part of the family Luteoviridae and is related to the Beet Chlorosis virus (BChV) and Beet Western Yellow virus (BWYV). Although it is common in Europe the disease has not affected agricultural production in the USA, as no traces have been found. The symptoms include interveinal and uniform yellowing of leaf tissue with the thickening and brittleness of older leaves, which resembles both BWYV and BChV symptoms (Russel, 1962). BMYV is an aphid transmitted virus through the vector M. persicae. Studies by the Plant Breeding Institute (PBI) showed that unlike the beet yellows virus (BYV) it is a true persistent virus. They examined the transmissions, results of which showed that successful transmissions occurred when M. persicae was fed for 24 hours, nevertheless they reached maximum transmission efficiency after three days of infection feeding (Russel, 1962). The PBI also proved that the BMYV persisted for long periods within M.persicae and a large proportion of aphids which had been fed on infected sugar beet for four days, managed to transmit the virus after nine days on Brassica pekinensis, which was supposed to be immune to BMYV. It also illustrates that unlike BYV aphids, aphids which had acquired BMYV could still transmit the virus after moulting without access to further sources of virus.
Fig 3 Aphid vector for BMYV Myzus persicae, BMYV is able to maintain persistent virulence within M. persicae. Luteoviruses are phloem-limited viruses which are transmitted by aphids in a nonpropagative persistant manner. Like other poleroviruses, BMYV is limited to the vascular tissue of its hosts and mechanical inoculation is only possible in mixed infections with umbraviruses (Mayo et al, 2000). The spread of the aphid is dependent on season, however due to the long lasting persistence it makes BMYV infection a serious problem.
The vector specificity of luteoviruses transmission suggests that specific cellular receptors in the aphid interact with the capsid. The major component of the luteovirus is the -22kDa polypeptide encoded by the open reading frames (ORF). BMYV has a genome organization with six large ORF located on a single-stranded positive-sense RNA. The ORFs are arranged in a 5' gene cluster (ORF0-2) and a 3' gene cluster (ORF3-5) (Guilley et al, 1995). ORF3-5 expression involves at least one sub-genic RNA.
Resistance Mechanisms
During the co-evolution of plants and their pathogens, the pathogens developed a wide variety of strategies to infect and exploit their hosts. In response to this pressure, plants reacted by deploying a range of defence mechanisms. Resistance to a pathogen is often accompanied by a response known as the hyper-sensitive reaction which is the rapid, localized death of cells at the infection site. In the most documented systems the occurrence of the hypersensitive reaction depends on the possession by the plant and invader of corresponding resistence (R) and a virulence (Avr) genes, also known as gene for gene interaction (Kombrink and Schmelzer 2001). In gene for gene, interactions involving viruses, viral gene products were identified as elicitors, capable of triggering the hypersensitive response include replication proteins, viral capsid proteins and viral movement proteins (Erickson et al 1999, Culver et al, 1994). Acquired resistance has been best characterised within tobacco and cucumber. During the 1960s it was demonstrated that infection with tobacco mosaic virus can cause tobacco to become resistant to diverse viral pathogens. This was termed systemic acquired resistance (SAR) which is proven to be effective against fungal and bacterial pathogens.
Arabidopsis thaliana has been used to identify resistance to BMYV. It is used as it has a small genome, rapid life cycle, high transformation efficiency with a completely sequenced genome and powerful reverse and forward genetics. It belongs to the Brassicaceae or Crucifer family, which includes the genera Arabis, Brassica and Cardamine. It is favoured for its small genome size (114.5/125Mb), extensive genetic physical maps of all 5 chromosomes, its rapid cycle and its prolific seed production and easy cultivations to produce polyploidization (Chen et al, 2000). The Arabidopsis thaliana provides a powerful genetic and genomic resource for elucidating mechanisms of gene and genome duplication.
High-throughput sequencing has generated abundant information on DNA sequences for the genomes of various plant species. This included the completion of the draft of Arabidopsis thaliana in 2000 (Chen et al, 2000). Previous expressed sequence tags from other important crop species have been mapped and generated to produce biotechnological tools and have annotated thousands of sequences as functional genes. The task of bridging this DNA sequence information with particular phenotypes relies on molecular markers (Chen et al, 2000). This is where this project will be focused by using an efficient PCR based SSR (Simple sequence repeats) technique to utilise polymorphic markers around the A. thaliana genome to identify ones linked to the BMYV resistance gene.
Microsatellite Analysis
Molecular markers linked to resistance genes are useful to follow genes in breeding programmes.
To locate resistance loci, they must be mapped relative to other markers. Mapping of genes in sugar beet lags behind mapping in other crops, e.g. the cereals. Before the advent of molecular markers, disease resistance gene mapping in sugar beet was slow and difficult and only one such gene, C for curly top resistance, Owen and Ryser, (1942) was mapped. The reasons for this slow progress in sugar beet were:
Sugar beet contains relatively few morphological markers that can be used for mapping (Francis et al, 2000).
Most sugar beet pathogens do not exist as physiological traits that can elicit trait-specific resistance responses in the host; therefore probably few gene-for-gene interactions exist that can be easily scored in a mapping population (Francis et al, 2000).
Resistance to many diseases appears to be controlled by one or more quantitative trait loci (QTLs) and these cannot be mapped without molecular markers (Francis et al, 2000).
The mapping of F2 populations with 3:1 segregation ratios is difficult to breed into sugar beet as in general it is an allogamous species and homozygous parents can only be produced if the self-fertility gene is present in the material, otherwise doubled haploids must be developed (Francis et al, 2000).
Microsatellites are tandem repeats of DNA sequences of only a few base pairs (1-6 bp) in length, the most abundant being the dinucleotide repeats. It was termed by Litt and Luty (REF?) to characterize the simple sequence stretches amplified by PCR (polymerase chain reaction) (Gupta et al, 1996). They are abundant and occur frequently and randomly in all eukaryotic nuclear DNAs examined (Gupta et al, 1996). Their frequencies vary significantly among different organisms (Wang et al, 1994). The length differences are attributed to the variation in the number of repeat units at a particular SSR locus, caused by slippage during replication (Gupta et al, 1996).
The benefits of using microsatellites provide the following:
They provide alleles that exist in a population and the level of heterozygosity is extremely high.
The markers are co-dominant (contribution of both alleles visible in phenotype).
The markers are inherited in a Mendelian fashion and thus for our investigation can be used for linkage analysis (Gupta and Varshney, 1999).
The frequencies of microsatellites have been examined in chloroplast genomes, since complete or partial sequences of chloroplast genomes are now known from several plant systems. These include rice, tobacco and maize (Gupta and Varshney, 1999) and they predominantly included short to medium sized stretches of mononucleotide sequences. It was shown that within these plant systems while fluorescent in situ hybridization suggested apparent clustering of microsatellites, genetic mapping in several cases demonstrates uniformly distribution throughout genomes (Gupta and Varshney, 1999).
DNA polymorphisms are detected by PCR by two methods. They either target individual loci using specific primers bordering the microsatellites (Gupta et al, 1996), or by using primers as synthetic oligonucleotides which are complimentary to a microsatellite motif randomly distributed throughout the genome (McCouch et al, 1997). Both assays typically carried a high information content and had been used for mapping and gene tagging (McCouch et al, 1997). The investigation will be using SSLP (simple sequence length polymorphisms) as it moreover allows restriction enzymes to be used to resolve digested CAPS (cleaved amplified polymorphisms), which has been previously used on tomatoes and barley (Davila et al, 1999).
AIM
So far within the investigation of BMYV a susceptible gene has been found within the Col-) ecotype of A. thaliana plant which has been crossed with a resistant ecotype Sna-1. The aim of this project was to investigate polymorphisms in the mutated Arapidobsis thaliana, Columbia and Sna-1. The purpose being to discover a marker to target the resistance gene (s) for Beet Mild Yellows Virus, in Arabidopsis thaliana. To do so various markers from chromosomes 1-5 were used to search for the resistant region of each chromosome.
METHOD
Inoculation:
The F2 generation of Arabidopsis thaliana was inoculated with BMYV infected Myzus persicae. This produced two plants (although not proven), Sna-1 and Col-0. The Sna-1 contained alleles BMYV-ss and had shown mutational resistance. The other Arabidopsis thaliana is thought to show susceptibility towards BMYV and therefore could contain the alleles SS as these were thought to be of a dominant phenotype. Sna-1 and Col-0 were bred to form the F1 generation which self fertilised to form F2 generation. The F2 plants will be either Ss, sS, or SS and susceptible or ss and resistant therefore there is a 3:1 ratio.
DNA extraction:
The Arabidopsis leaves were added to 700µl of Extraction Buffer (100mM Tris pH 7.5, 500mM NaCl, 50mM EDTA, 1.5% SDS and 0.1% β-mercaptoethanol) were added to a 2.5ml microfuge tube and then ground using a blue homogeniser until the leaf product had dissolved. These tubes were then incubated for 12 minutes at 65â°C. Continuing this, tubes then centrifuged at 13,000rpm for 8 minutes. 700µl of the supernatant was placed in clean microfuge tubes and 700 µl of phenol: chloroform was added and the tubes were further centrifuged at 13,000 rpm for 8 minutes. Next 540µl was used to clean the microfuge tubes and was added to 60µl of 3 M NaAc pH 4.8 with 600µl of isopropanol mix and incubated for 2 hours at -20â°C. The tubes were then centrifuged for 15 minutes at 13,000 rpm, the supernatant was removed and the remaining pellet was washed with 80% ethanol. The ethanol was afterwards removed and the pellet was re-suspended in 50µl sterile distilled water.
PCR Mixture
The PCR methodology was used under standard procedures set by PROMEGA using GoTaq Flexi. For one reaction mix the following PCR mixture shown in table 1 was added to 2.5ml microfuge tubes. The DNA polymerase was maintained in the freezer and added after the DNA preps to prevent early digestion and denaturing of the enzyme. The concentrations remained the same throughout for all the primers apart from G3883 on Chromosome 4 which had used 1.5MgCl2 (according to the special condition required through www.arabidopsis.org).
Table 1 List of reaction components used to make a PCR product.
Component
Final Volume
5 x Polymerase Green Go Taq Buffer
4µl
20mM dNTPs
0.2µl
Forward Primer (10µM)
1µl
Reverse Primer (10µM)
1µl
Go Taq Flexi Polymerase
0.1µl
MgCl2
1.2µl
Sterile Distilled Water
11.5µl
The PCR cycle
Table 2 The PCR cycle
Step
Time Seconds
Temperature â°C
A
30
94
B
30
55
C
30
72
D
Repeat from Step 'A' 34 times
E
300
72
Table 3 A list of the Microsatellite Markers from Chromosome 1-5 and the region in which they target Columbia
Microsatellite Marker
Chromosome Number
cM
Mbp
Col-0 length
NGA 111
1
115.55
26.69
111
NGA 248
1
42.17
23.45
143
NGA 128
1
83.32
20.22
180
ATH ANTPASE
1
117.86
28.53
85
CIW 2
2
76.8
1.19
105
CIW 3
2
53.76
6.4
230
NGA 1126
2
50.65
4.5
191
BIO 2
2
67.0
18.01
141
NGA 32
3
5.87
0.44
260
NGA 172
3
9.91
0.79
162
NGA 162
3
20.56
4.61
107
NGA 126
3
16.35
3.65
119
NGA 6
3
86.41
23.03
143
NGA 12
4
22.92
6.39
247
NGA 8
4
25.56
5.63
154
DET 1
4
31.44
6.35
DHS1
4
108.54
18.53
NGA 76
5
68.4
10.42
231
CA 72
5
29.6
4.25
124
NGA 225
5
14.31
1.51
119
NGA158
5
18.12
1.69
108
NGA 106
5
33.357
5.635
157
Restriction Enzymes
To G4539 and G3883 restriction enzymes were added for digestion after the PCR cycle. The restriction enzyme HindIII was used for G3883 and G4539 was tested using both HindIII and RsaI.
1µl from each restriction enzyme was added to the PCR tubes containing the DNA preps. This was then incubated at 37â°C overnight.
Microsatellite Analysis
The PCR reactions were used with the microsatellite markers in table 3. The primers were from chromosomes 1-5 and some included restriction enzymes to create a clear polymorphism of the F2 progeny, to enable clear distinction of the heterozygous individuals.
Table 4 shows the lengths of each chromosome 1-5.
Chromosome Number
Length (cM)
Length (mb)
1
135
29.2
2
97
17.5
3
101
23.6
4
125
22.2
5
139
26.2
Fig 4 shows a chromosomal map of the targeted loci for the available SSR microsatellite markers on each chromosome 1-5. This gives an indication of which markers to use to ensure each region of the chromosome is targeted.
Gel Electrophoresis
The agarose was tested at both 1.4% and 2.0% however 1.4% produced the best results. This consisted of 1.4g of agarose mixed with 100ml of TAE Buffer (40mM Tris-acetate pH 7.8 1mM EDTA pH 8) solution. 3µl of Ethidium Bromide was added after being melted and left to set in a gel tray containing the comb. After this was set TAE buffer was added and a further 3µl of Ethidium Bromide. The gel was then left to run at 110V for 70 minutes in a twenty lane gel and for 45 minutes in a 10 lane gel. Chi squared was afterwards calculated on each gel image to determine whether it had a 1:2:1 ratio or linkage.
RESULTS:
Non-targeted SSR analysis
SSR microsatellite markers were analysed that are located on chromosomes 1-5. The DNA preps were used on Sna-s-BMYV, Col-S-BMYV and the F1 plant for each marker to determine the polymorphism.
Fig 5 shows col, Sna-1 and F1s tested on various SSR primers from each chromosome. From using the previous chromosome map it was possible to pick 3 primers which target different regions of the chromosome. Chromosome 5 lane 1-3 is NGA 106 (Col, sna-1 and F1). Lane 3-6 (col, sna, F1) was CA 72 from chromosome 5. Lane 6-9 was NGA 76. Chromosome 4 was NGA 8 (lane 1-3), NGA 12 (lane 4-6) and DHS1(lane 7-9) of which the latter showed very little polymorphism. Chromosome 3 used NGA 162 (lane 1-3), NGA 6 (lane 4-6) and NGA 172 (lane 7-9). Chromosome 2 was CIW3 (lane 1-3), CIW 2 (lane 4-6)and NGA 1126,. Chromosome 1 used NGA 111, NGA 128 and finally NGA 248.
The chromosome 5 marker NGA 106 showed an approximately 33 base pair difference between Col and Sna-1 alleles and was easily scored (Figure ref). From this it was possible to select the polymorphisms from each chromosome and then test it on the DNA from the F2 progeny. This will allow a detection of segregation distortion and a deviation of the observed genotypic frequencies from the expected Mendelian ratios (1:2:1).
Table 5 shows the results for each microsatellite marker shown in Col-0 and Sna-1.
Microsatellite Marker
Chromosome Number
Polymorphism in Col and Sna-1
Col-0 allele
(bp)
Sna-1 allele
(bp)
NGA 111
1
Yes
111
103
NGA 248
1
Yes
143
150
NGA 128
1
Yes
180
175
CIW 2
2
No
105
105
CIW 3
2
Yes
230
210
BIO 2
2
Yes
141
151
NGA 172
3
Yes
162
186
NGA 162
3
Yes
107
115
NGA 6
3
Yes
143
119
NGA 12
4
No
247
--
NGA 8
4
No
154
--
DHS1
4
No
--
--
NGA 76
5
Yes
231
253
CA 72
5
Yes
124
120
NGA 106
5
Yes
157
135
Microsatellite Analysis
The microsatellite analysis initially proved unsuccessful in demonstrating specific linkage of SSRs to the resistance gene in Sna-1. The microsatellite markers chosen identified that the F1 generation was a genuine cross and both Col and Sna-1 bands were present in each lane (fig. 6). This was also determined in the F2 progeny which produced 1:2:1 ratios with homozygous and heterozygous alleles.
Chromosome 5 Gel images
The marker NGA 106 produced the best polymorphism and showed clear distinction between the two bands and produced clear heterozygote bands in both the F1 and F2 generation.
Fig 6 shows the microsatellite gel image for NGA 106 with Col, Sna-1 and het alleles. Lane 1 shows a 1kb marker, lane 2-4 shows Col, Sna-1 and F1 with clear polymorphism. Lane 5-20 contain the F2 generation. Col homozygous bands present in (F2-72R, F2-76R, F2-29R, F2-126R). Sna-1 homozygous bands present in (F2-131R, F2-43R, F2-78R, F2-127R). Heterozygous bands present in (F2-179R, F2-186R, F2-42R, F2-139R, F2-129R, F2-25R, F2-102R)
The gel image from fig 6 shows that through DNA extraction from plants with Col and Sna-1 and the F1 generation had adequate amounts of DNA. From this it was possible to put the DNA under PCR and amplify the relevant strands (needs to be more clearly written). It is clear that the F1 generation produced true crosses and it had successfully self-fertilised. The Col bands appeared around 135 and the Sna-1 bands occurred at 157.
There are fifteen samples of which four are Columbia and 4 Sna-1 with a further seven producing heterozygous results. Chi squared produced a 1:2:1 ratio so no linkage was detected.
When compared to other markers we can clearly see to the extent the clarity of the polymorphism. Fig 6 shows no polymorphism from NGA 172 from chromosome 3 as is shown in the gel image.
Fig 7 shows the PCR gel electrophoresis image produced from NGA 172. Lane 2-4 Col, Sna, F1. Lane 5-19 shows the F2 plants. Col-0 was seen in bands (F2-78R, F2-42R, F2-129R). Sna-1 homozygous bands present in (F2-179R, F2-168R, F2-76R, F2-127R). Heterozygous bands were seen in (F2-131R, F2-43R, F2-78R, F2-139R, F2-72R, F2-76R, F2-126R, F2-197R).
The result from fig 7 makes scoring of the result unreliable, however in this case it was just about possible. For other results which produced unreliable results they were run on longer gels, such as CIW3 from chromosome 2. This was run on a 20 lane gel for a longer period of time to separate the bands clearly.
Fig 8 shows gel image from CIW3 from chromosome2 and shows clear polymorphism. Lane 1 is the 1kb marker lane 2-4 shows col, sna-1, F1. Lane 5-20 shows the F2 generation. Col-0 homozygous bands were present in plants (F2-197R, F2-131R, F2-78R). Sna-1 bands were present in (F2-168R, F2-42R, F2-76R, F2-179R, F2-102R). Heterozygous bands were present in plants (F2-43R, F2-25R, F2-127R, F2-129R, F2-29R, F2-72R, F2-126R, F2-168, F2-43, F2 25, F2 42, F2 127, F2 197, F2 76, F2 131, F2 179, F2 102, F2 129, F2 29, F2 72, F2 78, F2 126, F2 139R).
Once every available marker had been used to target regions of chromosome 1-5, they were scored and the chi-squared was calculated to determine whether it showed a 1:2:1 ratio or segregation distortion, towards the resistance plant Sna-1. The Chi square test will establish whether the observed numbers of phenotypes deviate significantly from those expected in case of independent assortment.
Table 6 Chi-squared Analysis of each Chromosomal Marker of Sna-1 (+/+), Col-0 (-/-) and F1 (+/-) (P value 5.991)
Marker
Chromosome
+/+
-/-
+/-
Chi Value
Allele Ratio
NGA 111
1
5
6
4
0.7332
1:2:1
NGA 248
1
1
2
1
2.5
1:2:1
NGA 128
1
1
2
1
1.85
1:2:1
ANTH ATPASE
1
3
3
8
0.6
1:2:1
CIW 3
2
3
5
8
0.5
1:2:1
BIO 2
2
3
7
9
0.6
1:2:1
NGA 6
3
3
3
9
0.6
1:2:1
NGA 172
3
4
3
7
0.142
1:2:1
NGA 162
3
3
5
7
0.86626
1:2:1
NGA 8
4
4
2
7
0.1356
1:2:1
NGA 12
4
DHS1
4
NGA 106
5
4
4
7
0.998
1:2:1
NGA 76
5
4
4
7
0.0998
1:2:1
CA 72
5
3
3
8
0.6
1:2:1
After successfully scoring 15 SSRs on the F2 progeny, there was no linkage detected to the resistance in the Sna-1 ecotype. However after successful sequencing of AFLP markers linked to the susceptibility allele in Col-0 it was shown that the gene is actually located on Chr 4in the region 7533850-106182901bp. At the time the only available primer combination to markers in this region was G4539 which was on the region 1589139 - 1589703 bp on chromosome 4.
Fig 9 G4359 caps marker with restriction enzyme Hind111. Lane 1-3 old extracted Col-0, Sna-1, F1. Lane 4-5 new extracted Col-0 and Sna-1. This marker does not show adequate polymorphism.
Fig 10 G4539 caps marker from chromosome 4 with restriction enzyme RSA1. Lane 1-3 Col-0, Sna-1 and F1. Lane 4-5 New extracted Col-0 and Sna-1. Shows clear polymorphism between both bands, with visible bands on the F1 progeny.
After successfully detecting a restriction enzyme which cuts the DNA at a region producing clear polymorphism it was then used for a full analysis on the full F2 as shown in fig 11.
Microsatellite Analysis of F2 Progeny using G4539 + RSA 1
Fig 11 G4539 with RSA1 used on the full F2 DNA progeny. Two heterozygous plants were present in F2-103 and F2-114 progeny. The rest which were possible to score were Sna-0 bands.
Unfortunately the results produced poor images even after staining with further Ethidium Bromide. The procedure was also repeated with further incubation time for the restriction enzyme to digest, with no avail. Although it is clear to see that from the gel images that there is linkage to the Sna-1 resistance gene. It was possible to determine that 27 were homozygous for the Sna-1 allele 8 were heterozygous.
A further primer was needed to generate a genetic map and locate the region at which the primers were targeting chromosome 4. This was provided by the John Innes Centre as the primer CIW6 which targets Columbia at a region of 0.162kp and G3883 which target the region of Columbia at 1.4kb (???).
Figure 10: AGI Map for chromosome 4 as obtained from [http://arabidopsis.org/servlets/mapper?action=search&band=0&field=CIW6&map0=on&map1=off&map2=off&map3=off&map4=off&map5=off&map6=off&map7=off&option=selected]
The primer G3883 proved unsuccessful at producing any polymorphism however CIW6 showed clear polymorphisms and clear linkage to Sna-1 as shown in fig 10.
Fig 12 Shows a gel image of PCR with CIW6 primer from chromosome 4. Lane 1-3 Col-1, Sna-0 and F1, F2 25, F2 131, F2 168, F2 179, F2 43. The F2 plants F2-25, F2-131, F2 168 and F2 179 show linkage towards the Sna-1 region. This produces a corrupt 1:2:1 ratio.
Micosatellite Analysis on F2 progeny using CIW6 Marker on Chromosome 4
Fig 13 Full Microsatellite analysis of CIW6 on 46 F2 DNA preps with six heterozygous plants (F2-43, F2-93, F2-103, F2-114, F2-166, F2-221).
Position of the Primers on Chromosome 4
Table 7 The heterozygous recombinant alleles from both CIW6 and G4539 markers
F2 Plant
CIW6
G4539
43
H
-
93
H
-
103
H
H
114
H
H
166
H
-
221
H
-
Table 8 Total Progeny and phenotype for both CIW6 and G4539
Homology
CIW6
G4539 +RSA1
Col-0
1
0
Sna-1
40
23
Heterozygous
6
1
Recombination
7%
1%
Table 7 and 8 suggests CIW6 shows recombination of 7cM and G4539 shows a recombination of 1cM. The two heterozygous plants 103 and 114 when used with both G4539 and CIW6 both appear as recombinants, however G4539 is not conclusive as the scoring from the polymorphism could not be used.
Discussion
The purpose of this project was to continue previous research on the test crosses of Sna-1 and Col-0. The plants were selected for F2 progeny and the analysis was to detect linkage by defined SSR markers.
The investigation provided evidence pointing towards linkage of the resistance gene (s) for Beet Mild Yellows. The SSR polymorphism technique was used to detect polymorphism to confirm linkage. In the initial part of the experiment, clear polymorphism was not confirmed, however, on chromosome 5 which was targeted by the microsatellite marker, NGA 106. It produced distinct polymorphism that was exhibited at an almost 33bp difference between Col-0 and Sna-1 ecotypes. As was expected the marker, NGA 106, produced distinct heterozygous bands in both the F1 and F2 generation. Using all of the available primers it became apparent that successful 1:2:1 ratios were present in the F2 progeny. This confirms that Col-0 produces a single dominant gene and that the BMYV resistance is a single recessive trait.
After successful sequencing of AFLP markers from Col-0 a possible chromosomal region for location of the susceptibility gene was discovered on chromosome 4 in the region of 7533850-106182901bp and the marker CIW6 was established and developed, by the John Innes Centre. This proved successful and showed DNA fragments that were found to be linked to the resistance gene in Sna-1. This was confirmed by calculating the number of heterozygous alleles and dividing it by the total number of heterozygous and homozygous alleles. The results indicate a 7cM recombination which gave an idea of the distance from the gene, however it required the use of another marker to measure the amount of recombinants present and to detect which side of BMYV-s-Sna-1 it targets.
The marker G4539, to which restriction enzyme Rsa1 digest was added in PCR, did not show a 1:2:1 ratio and produced linked Sna-1 bands. However, it did not produce adequate results to map the gene. To locate this marker in correlation to BMYV-ss-Sna-1 it needed to be flanked by markers mi260 9076497 - 9077409 bp. Perhaps more focus put through better staining of the fragments. Bowers et al (1996) suggests that silver staining is a more reliable method than automated fluorescence methods. It was found that while SSR amplification maybe analyzed by ethidium bromide using agarose gels, the allele size cannot be reliably estimated by this method and small size differences cannot be resolved as they can in acrylamide sequencing gels (Bowers et al, 1996). Allele size difference and clarity is determined by 1-2bp (Thomas et al, 1994).
Although from the fig 11, it was possible to score 2 recombinants, which could suggest this marker is closer to the resistant gene.. This remains to be confirmed with the use of other markers to flank this marker in respect to BMYV-s-Sna-1.
The two markers did prove that they were present on the same side of the gene along chromosome 4, which eliminates the possibility that they were on different sides of the resistance gene. This is because the two recombinant plants F2-103R and F2-114R both appeared as heterozygous. If one ecotype had shown a heterozygous and the other a homozygous trait this would have confirmed that they were on separate sides of the resistant gene.
The two possible scenarios are mapped as follows:
Fig 14. The two markers linked to the BMYV-ss region mapped using Arabidopsis thaliana. Two possible outcomes were obtained, however further markers must be used to gain the exact loci position.
This information could prove valuable for combining sources of resistance and to study whether resistance genes are alleles or situated at different loci along chromosome 4???.
Similar studies have proved successful such as that for Rhizomania which is formed through the virus necrotic yellow vein virus (BNYN). Families for resistance to rhizomania were discovered by crossing resistant and susceptible and inoculating them. Barzen et al (1997) then used molecular markers to combine sources of resistance to study whether resistance genes are alleles or whether they are situated at different loci. This proved successful as markers found to be linked to Rhizomania resistance to show resistance in B. vulgaris sub-species and in the commercial hybrid 'Golf' indicating the possible existence of identical loci in these accessions.
In conjunction to the relevance with sugar beet most mapping has been based upon RFLP and RAPD markers on the nine linkage groups of B. Vulgaris (Schondelmaler et al, 1995). Until now only a few have combined different mapping techniques and furthermore most linkage maps do not establish the relationship between observed linkage and actual chromosomes (Schumacher et al, 1997). For the first time each of the linkage groups could unequivocally be assigned to one of the sugar beet chromosomes using SSR (). This had been based upon correlations between the karyotype of B.vulgaris chromosomes with all published linkage groups, and on the linkage relations with respect to mutants and molecular markers.
The application of marker-assisted selection within breeding programs for virus is as yet still limited, due to the lack of co-ordinated markers associated with BMYV resistant genes. The successful application of marker-assisted selection for virus resistence will be dependent on access to easily applied markers with close linkage to resistance genes. Previous markers have been developed for Ry (Hamalainen et al, 1997; Kasai et al, 2000) and for PVS the major resistance gene (Marczewki et al, 2002) and for the Rx PVX resistance gene (Bendahmane et al, 1997; De Jong et, 1997).
There are considerable variations in host ranges and serological reactions which have been shown to exist between members of the luteoviruses (Russel, 1965). BMYV and BWYV were considered to be strains of the same virus (Casper, 1988), even though they fed upon variable host ranges. It wasn't until the complete nucleotide sequence had been determined for BMYV previously by Guilley et al (1995) that it was discovered that BMYV should be considered a distinct virus rather than a strain of BWYV (Beet Western Yellows Virus). The nucleotide sequence of the genomic RNA of BMYV consists of 5722 nucleotides and six long open reading frames which conform to the arrangement characteristic if subgroup 2 luteoviruses (Guilley et al, 1995). Comparative analysis of complete genome sequences from BWYV and BMYV showed that BMYV is indeed a recombinant between two poleoviruses.
Little is currently known about the field strains in the UK, although studies by Brooms Barn has identified BMYV-BB-NC which infects sugar beet but not C bursa-pastoris or M perfoliata (Stevens et al, 1994). Also a strain BYDV-PAV-IL-1 is used to identify BMYV in winged aphids migrating into sugar beet crops (Smith et al, 1991). Although it has also been proven that BYDV-PAV-IL-1 does not detect the resistant strain BMYV-BB-NC, suggesting that BYDV-PAV-IL-1 underestimates the number of virus carrying aphids and infected plants. Also recently a monoclonal antibody was produced, MAFF-24, which showed considerable increase in sensitivity for detecting BMYV. This antibody is now used throughout the UK however it cannot distinguish between BMYV and BWYV (Smith et al, 1995).
With the growing number of polymorphic SSR markers available for targeting susceptible and resistant regions this could allow the removal of specific sequences for breeding. It also enables new strains of viruses to be implicated such as the new strain of Rhizomania which is now overcome the resistant variety. Research from Brooms Barn is now targeting this new strain with previous markers. To build up an archive knowledge of polymorphic markers is crucial in the battle against crop pathogens.
