Genetically Marking the susceptible Gene for Beet Mild Yellows in Arabidopsis thaliana

The sugar beet pathogen Beet Mild Yellows Virus was inoculated using the aphid Myzus persicae into the model plant, Arabidopsis thaliana. This produced two plants one which expressed possible resistance in the form of BMYV-ss-Sna-1 and the other, susceptibility BMYV-SS-Col-0. After crossing Col-0 and Sna-1 to create an F1, the offspring was self fertilised to create an F2 generation which exhibited a 31 ratio of susceptibility to resistance. The susceptibility locus, was investigated by using a technique called 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 analysis can be done on the resistance gene in Arabidopsis thaliana.

Sugar beet background
Sugar beet is a crop of immense economic value as it is used in the manufacture of sugar and is used as animal fodder. It is also a major component in the manufacture of Bio-fuel.  Even though the British beet sugar industry began during the early 1900s, the first extraction of sugar, however, was demonstrated by the Prussian chemist Andreas Sigismund Marggraf, in 1747 a method in which alcohol was used to extract sugar from beets. Yet this method did not go into industrial scale production, until after the blockade of continental ports which occurred during the Napoleonic wars and 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. This led to the first sugar beet factory in Prussia in 1801 which contained at the time only about 4 sugar incorporated into the beet.

In the UK, the beet industry began during the 1900s with the first factory being built by the Dutch of Cantley in Norfolk in 1912. This then shot up to 17 factories during the 1920s and the crops were processed by 13 autonomous companies. In 1936, those factories were amalgamated by the Sugar Industry Act to from 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 is contracted with 4000 farmers and between September and March, as much as seven and a half million tonnes of sugar beet are delivered to the factories. In one factory alone, 14000 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. It also provides other vital substances through the production of sugar beet such as recycled stones for building, lime for soil conditioning and soil for landscaping. They are also used in combined heat  power plants to generate and export electricity enough for 350,000 people and use the combustion gases to grow 80 million tomatoes. More recently and almost more importantly, they are being used to as alternatives to fossil fuels which are fast depleting. British Sugar has invested in the UKs first bio-ethanol plant, producing 70 million litres of renewable fuel through the production of Sugar Beet, to help prevent a global economic crisis (British Sugar n.d.)

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. During its first growing season it is used for commercial harvesting and can produce as large as 2kg tubules which contain 15-20 sucrose by weight. During its second season the nutrients in the roots are used to produce flowers and seeds in cold weather, even though the process cannot take place in extreme cold conditions due to the crops poor frost resistance. During its seedling stage it is a poor competitor with weeds and can also be damaged by Heterodera schactii, a pathogenic nematode in beet. Sugar beet also suffers from viral infection which inhibits the plant 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 found in the south (Smith, 1987). It causes beet leaves to 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). It has a large effect on the yield if the virus infects the plant during early growth, decreasing the overall yield by 30. This has been estimated to have cost the national sugar industry 24,700 tyear which is the equivalent of 1.8 of the countries yield and a staggering 5.5 million.      

The BMYV virus 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). It is common in Europe however as of yet has not affected the 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 showed that unlike the beet yellows virus (BYV) it is a true persistent virus. They examined the transmissions and results showed that successful transmissions occurred when M. persicae was fed for 24hrs, however they reached maximum transmission efficiency after three days of infection feeding (Russel, 1962).  They 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 9 days on Brassica pekinensis, which was supposed to be immune to BMYV. It was also shown 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 leastone sub-genic RNA.

Resistant 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 countered 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 resistance (R) and avirulence (Avr) genes, also known as gene for gene interaction (Kombrink and Schmelzer 2001). In gene for gene interactions involving viruses, viral gene products 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 with in 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 has been used to map BMYV and recognise the resistant genes. 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 (114.5125Mb), 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 model polyploidy model provides a powerful genetic and genomic resource for elucidating mechanisms of gene and genome duplication. Genome-wide changes in gene expression can be comparable with changes in genome organisation as well as a chromatin structure in auto and allopolyploids which provide a mechanistic view of polyploidy effects on the regulation of homologous gene. (Chen et al, 2000)

High-throughput sequencing has generated abundant information on DNA sequences for the genomics of various plant species. This included the completion of the model draft of Arabidopsis thaliana in 2000 (Chen et al, 2000). This also includes other express 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 my project will be focusing by using an efficient PCR based AFLP (Amplified Fragment Length Polymorphism) technique to generate polymorphic markers around targeted putative resistant gene sequences against BMYV.

Microsatellite Analysis
Molecular markers linked to resistant genes are useful to facilitate genes in breeding materials.
To locate resistance loci, they must be mapped relative to other markers. Mapping of genes in sugar beet is somewhat 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 (1-6bp) in length, the most abundant being the dinucleotide repeats. It was termed by Litt and Luty (Cregan 1992) 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 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 fluorescence 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 also 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).

So far within the investigation of BMYV a susceptible gene has been found within a mutated Col plant which has been crossed with a possible resistant gene from a Sna-1 plant. The aim of my project was to investigate polymorphisms in the mutated Arapidobsis thalium, Columbia and Sna-1. The purpose was also to discover a marker to target the susceptible gene (S) for Beet Mild Yellows Virus, in Arabidopsis thalieum. To do so various markers from chromosomes 1-5 were used to search for the resistant region of each chromosome. This is because the susceptible region is dominant SS and therefore has a 75 region of the chromosome, whereas the resistant gene ss will give a more accurate region as it is much smaller.

Arabidopsis thaliana was previously 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 Ss, sS, or SS and susceptible or ss and resistant therefore there is a 31 ratio.

DNA extraction
The Arabidopsis leaves were added to 700l of Extraction Buffer (100mM Tris pH 7.5, 500mM NaCl, 50mM EDTA, 1.5 SDS and 0.1 -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 mins at 65p C. The tubes were then centrifuged at 13,000rpm for 8 minutes. 700l of the supernatant was placed in clean microfuge tubes and 700 l of phenol chloroform was added and the tubes were further centrifuged at 13,000 rpm for 8 minutes. Then 540l was used to clean the microfuge tubes and was added to 60l of 3 M NaAc pH 4.8 with 600l of isopropanol mix and incubated for 2 hours at -20p C. The tubes were then centrifuged for 15 mins at 13,000 rpm, the supernatant was removed and the remaining pellet was washed with 80 ethanol. The ethanol was later  drained and the pellet was re-suspended in 50l sterile distilled water.

Random Frequency Length Polymorphisms
This approach amplified and performed using a 5anchored SSL primer. The amplified products resolved length polymorphism that presented either the SSL target site itself or at the associated sequence between the binding sites of the primers. This also allowed the amplified products to be digested with restriction enzymes to gain the genetic map if linkage occurred.

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 kept 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 in TAIR)

Table 1 List of reaction components used to make a PCR product.
ComponentFinal Volume5 x Polymerase Green Go Taq Buffer4l20mM dNTPs0.2lForward Primer (10M)1lReverse Primer (10M)1lGo Taq Flexi Polymerase0.1lMgCl21.2lSterile Distilled Water11.5l
The PCR cycle
Table 2 The PCR cycle
StepTime SecondsTemperature CA3094B3055C3072DRepeat from Step A 34 timesE30072

Table 3 A list of the Microsatellite Markers from Chromosome 1-5 and the region in which they target Columbia
Microsatellite MarkerChromosome NumbercMMbpCol-0 lengthNGA 1111115.5526.69111NGA 248142.1723.45143NGA 128183.3220.22180ATH ANTPASE1117.8628.5385CIW 221.19105CIW 326.4230NGA 1126250.65191BIO 2267.018.01141NGA 3235.870.44260NGA 17239.910.79162NGA 162320.564.61107NGA 126316.353.65119NGA 6386.4123.03143NGA 12422.926.39247NGA 8425.565.63154DET 1431.446.35DHS14108.5418.53NGA 76568.410.42231CA 72529.64.25124NGA 225514.311.51119NGA158518.121.69108NGA 106533.3575.635157

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 which cuts at 300bp.

1l from each restriction enzyme was added to the PCR tubes containing the DNA preps. This was then incubated at 37C 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 restrictive enzymes to create a clear polymorphism of the F2 progeny, to enable clear distinction of the heterozygous alleles.

Table 4 shows the lengths of each chromosome 1-5
Chromosome NumberLength (cM)Length (mb)113529.229717.5310123.6412522.2513926.2

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
Agarose gels were made to show electrophoresis on the separate DNA strands. The agarose was tested at both 1.4 and 2.0 but 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. This was melted and 3l of Ethidium Bromide was added and left to set in a gel tray containing the comb. After this was set TAE buffer was added and a further 3l 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 then calculated on each gel image to determine whether it had a 121 ratio or linkage.

The PHD student collected plant material from a all resistant and all susceptible F2 plants. A DNA extraction was performed to gain DNA from resistant and susceptible plants. This was then cut up the DNA with restriction enzymes Mse1 and Pst1. It was then radio labelled with the Pst primer and and further un-labelled with Mse primer. It was then put through PCR which produced various fragments of different sizes. They were then run on a Urea gel, and left to expose a film. Once the film and had identified polymorphisms within the DNA, it was sequenced out of the gel and resuspended in water. This was then sent off to the John Innes Centre for sequencing.

Polymorphisms of the Test Markers for each Chromosome

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 microsatellite markers varied in the amount of polymorphism they expressed however it was shown in chromosome 5 to have the greatest polymorphism in NGA 106 which showed approximately a 33 base pair difference between Col and Sna-1. From this it was possible to select those which had shown the greatest polymorphism from each chromosome and then test it on the DNA from the F2 progeny from the F1 plant crosses.

Table 5 shows each microsatellite marker and the results of the region they produced the bands Col-0 and Sna-1.
Microsatellite MarkerChromosome NumberPolymorphism in Col and Sna-1Col-0 length
(bp)Sna-1 length
(bp)NGA 1111Yes111103NGA 2481Yes143150NGA 1281Yes180175CIW 22No105105CIW 32Yes230210BIO 22Yes141151NGA 1723Yes162186NGA 1623Yes107115NGA 63Yes143119NGA 124No247--NGA 84No154--DHS14No----NGA 765Yes231253CA 725Yes124120NGA 1065Yes157135

Microsatellite Analysis
The microsatellite analysis initially proved unsuccessful in finding any specific linkage of Sna-1 however it did give good polymorphic images. 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. This was also determined in the F2 progeny which produced 121 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.
Figure 6 Before sequencing NGA 106 was used on chromosome 5 shows no linkage i.e. 121    ratio. The key gives the source in the lanes of the gel.

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 to try and map the chromosomes after amplifying. From this it was possible to put the DNA under PCR and amplify the relevant strands. It is clear that the F1 generation produced true crosses and had not just self-fertilised and the same can be said for the F2 generation. The Col bands appeared around 135 and the Sna-1 bands occurred at 157, according to fig 6. Unfortunately, results from areas like lane 10 where the image is not clear is due to poor absorption of the Ethidium Bromide, however it was still possible to score this result.

There are fifteen samples of which four are Columbia and 4 Sna-1 with a further seven producing heterozygous results. Chi squared produced a 121 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 121 ratio or segregation distortion, towards the resistant 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 (-)
MarkerChromosome -- -Chi ValueAllele RatioNGA 11115640.7332121NGA 24811212.5121NGA 12811211.85121ANTHATPASE13380.6121CIW 323580.5121BIO 223790.6121NGA 633390.6121NGA 17234370.142121NGA 16233570.86626121NGA 84NGA 124DHS14NGA 10654470.998121NGA 7654470.0998121CA 7253380.6121

After unsuccessfully scoring 15 primers with the F2 DNA progeny which produced the most DNA, there was no successful linkage found toward the Sna-1 ecotype. However after successful sequencing of the susceptible and resistant DNA from Col-0 and Sna-1 plant ecotypes a possible resistant region was discovered on chromosome 4 in the region 7533850-106182901bp. At the time the only available primers were G4539 which was tested on the region of 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 producing a restriction enzyme which cuts the DNA at a region producing clear polymorphism was then for a full microsatellite analysis used on the full F2 generation progeny, as shown in fig 8.

Microsatellite Analysis of F2 Progeny using G4539  RSA 1
Fig 11 G4539 with RSA1 used on the full F2 DNA progeny. 1 shows Lane 2-4 Col-0, Sna-1, F1. Lane 5-20 F2 29, F2 179, F2 76, F2 168, F2 225, F2 25, F2 45, F2 204, F2 166, F2 102, F2 127, F2 43, F2 93, F2 42, F2 72. Gel image 2 shows lane 2-4 Col-0, Sna-1 38, F2 129, F2 131, F2 173, F2 101, F2 37, F2 53, F2 115, F2 103, F2 139, F2 135, F 4, F2 7, F2 5, F2 159, F2 226. Gel image 3 shows F2 114, F2 126, F2 64, F2 82, F2 178, F2 182, F2 209, F2 197, F2 228, F2 152, F2 149, F2 66, F2 11, F2 12, F2 196

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 TAIR

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 121 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 PlantCIW6G453943H-93H-103HH114HH166H-221H-
Table 8 Total Progeny and phenotype for both CIW6 and G4539
HomologyCIW6G4539 RSA1Col-010Sna-14023Heterozygous61Recombination71
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.

The beet mild yellows virus (BMYV) causing Beet mild yellows disease in sugar beet has been a great cause of worry in agriculture. It is spread through aphids or Myzus persicae and causes premature yellowing of leaves and hence lessened rate of photosynthetic activity. This not only affects the sugar content of the plant, the leaves are rendered useless as fodder.

This project was conducted to inspire work on preventing this debilitating disease of the sugar beet crop and prevent the loss in economy caused by the BMY virus. Much work has been done on viruses affecting other crops like the tobacco mosaic virus, cucumber mosaic virus. However, as has been shown earlier, sugar beet is an important contributor towards our economy and is a major component in the production of bio-fuel for the benefit of future generations, the study of diseases and their pathogens on this important crop requires careful study. Other diseases on sugar beet like beet curly top virus, beet necrotic yellow vein virus and beet yellows virus have also been studied extensively. However, our main aim was to focus on the Beet Mild Yellows Virus as this virus has also been a major hindrance towards effectively increasing the output of the sugar beet crop in a year.

The experiment conducted was to isolate two strains of the model plant Arabidopsis thaliana where one would show resistance and the other susceptibility towards the BMYV. This was in continuance with previous research performed by other scientists, on the test crosses of Sna-1 and Col-0. We wished to determine the susceptibility of BMYV by using aphid transmission to infect plants and generating and studying a self fertilized F2 generation. The investigation yielded one mutated Columbia plant that showed definite susceptibility towards the disease and one from Sna-1 that showed a possible resistant gene. 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. Thus, we carried out the crossing of these two parents. The F2 results pointed out the presence of susceptible gene in 31 ratio to the resistant ecotype proving the susceptible gene to be dominant against the resistant gene. Hence the genotypes of the progeny can be concluded to be of SS, Ss, sS (susceptible) and ss (resistant) variety.

The scope of this experiment also permitted us to find evidence pointing towards linkage of the susceptible gene (S) for Beet Mild Yellows Virus. We used various markers on the five chromosomes to locate the target gene. Amplified fragment length polymorphism technique was used to create clear polymorphism to confirm linkage. In the initial part of the experiment, clear polymorphism was not confirmed, however, on chromosome 5 which was acted upon by the microsatellite marker, NGA 106 distinct polymorphism was exhibited with an almost 33bp difference between Col-0 and Sna-1 ecotypes. This enabled us to compare the results from the F2 progenies with the chromosomes showing polymorphism. As was expected the marker, NGA 106, produced distinct heterozygous bands in both the F1 and F2 generation.

The primer NGA 106 an SSLP type marker used as PCR primer made to act on chromosome 5 with sequences TGCCCCATTTTGTTCTTCTC and GTTATGGAGTTTCTAGGGCACG (Name 2010) exhibited the most clear results on the full F2 generation. It was clear that F2 generation had two distinct clear bands on the agarose gel image, making it possible for clear scoring of the parent plants Sna-1 (ss), Col-0 (SS) as well as recombinants (Ss). Using all of the available primers it became apparent that successful 121 ratio was present in the F2 progeny.

After successful sequencing of the susceptible and resistant DNA from Col-0 and Sna-1 plant ecotypes a possible resistant region was discovered on chromosome 4 in the region of 7533850-106182901bp. We used SSLP with markers for specific regions to target the resistant area. The marker CIW6, provided by John Innes Centre, proved successful and showed distinct linkage to Sna-1. The results indicate a 7cM recombination which gives an idea of the distance from the gene however it requires 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 121 ratio. However, it also did not produce adequate results to map the chromosome.

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 available data, it appears that G4539 is slightly closer as it shows less recombination, it 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 resistant gene. This is because the two recombinant plants F2-103R and F2-114R both appeared as heterozygous. The two possible e 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 implies that when crossing over takes place in Col-0 and Sna-1, the sites for G4539 and CIW6 are inherited together and therefore show linkage. With the use of other markers to flank these sites we should be able to zero in on the site at which the resistant gene may be present. 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.  Unfortunately, due to lack of time we could not repeat the results with G4539 and RSA and hence could not predict its exact position on chromosome 4, however, if we pursue this experiment, whenever time permits, we should be able to score progenies of both plants and come out with clear results of heterozygote. As given in figure 10, the sites of CIW6 and G4539 are marked according to our estimates, which show that they act at close proximities and hence linkage is possible.

There have been other studies done on this particular disease but we wanted to determine for ourselves, which those sites are that contain the mutant resistant gene. The future prospect for this study is to achieve sugar beet plants that have been genetically modified to possess the resistant gene to overcome this viral infection.  Some of the work done on Arabidopsis in this field are sequencing and cloning of BMYV in pLitmus 29 and inserting the full-length capped RNA transcripts in Arabidopsis thaliana using electroporation. For this purpose the BMYV sequence had to be modified with DNA encoding jellyfish green fluorescent protein (GFP) at 3 end of P5 gene. The results showed up to 2 fluorescence. (Stevens  Vigano 2006).The progress made in this line is crucial to understand the properties of the virus and ways to overcome the disease.

Techniques used for routine detection of plant viruses
The techniques most commonly used to detect virus infection in plants are using enzyme labelled antibodies or amplification of viral genome by PCR or nucleic acid amplification techniques. However, molecular and serological techniques are fast becoming popular as the results are obtained in a very short time, in confined space without propagating the pathogens. Enzyme-linked immunosorbent assay or ELISA test and DAS ELISA (double antibody sandwich) are now being used to detect plant viruses. Virus particles get trapped by specific antibodies on the surface of wells during culture of the sap extracts. These are detected easily by enzyme-labelled antibodies that convert the substrate into brightly coloured pigments on the plates that are easily detected by the naked eye (Koenig 1981)

The beet mild yellows virus has a genome with six large open reading frames (ORFs) located on a single-stranded positive-sense RNA. The infection mostly remains localised in the vascular tissues. (Stephan  Maiss 2006)

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 wasnt 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 have 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 can now overcome the resistant variety. Research from Brooms Barn is now targeting this new strain with previous markers. To build up archive knowledge of polymorphic markers is crucial in the battle against crop pathogens.

Hence, our experiment with finding the linkage of the recessive resistant gene with a suitable marker is a step towards better understanding the disease. Our study will definitely be needed in future when the exact locus of the resistant gene is to be located. Other markers are needed to flank the loci, which was beyond the scope of our study. Thus, we have covered a significant portion in proving that the resistant gene does display linkage and it is my belief that we shall, very soon be able to carry our project to a new level.


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