Marker-assisted selection was performed on 605 potato progeny using PCR to screen for the late blight resistant gene RB. 55% of the population was removed, leaving 275 potatoes with the RB gene for further evaluation in the field. Field days and presentations were given to Wisconsin potato growers that increased their knowledge of the concepts used in marker-assisted selection. Specifically, growers increased their knowledge about resistance genes, marker-assisted selection and how the RB gene will be beneficial to them in the future.
Potato late blight, a disease caused by the oomycete pathogen Phytophthora infestans, is one of the world’s most devastating plant diseases. None of the currently grown potato cultivars in the United States has adequate resistance to late blight. In major epidemic years fungicides have to be applied weekly to control the disease. The cost for fungicide is easily in the range of $4-8 million per season in Wisconsin alone plus at least $4 million to cover the cost of application (Stevenson, 2003, The Badger Common’tater 55 (9): 10-11). $70-140 million is spent for fungicide against late blight in the U.S. Such heavy fungicide applications lead to significantly less profitable potato production and also have a negative environmental impact. Utilization of late blight resistant varieties is the most cost-effective and environmentally friendly method to control late blight. Therefore, it is appropriate that we use faster breeding methods to produce a potato that will have adequate protection against late blight and have a positive impact on the environment.
At least 11 resistance (R) genes originated from Solanum demissum were incorporated into numerous potato varieties. However, these R genes confer race-specific resistance. Potato cultivars possessing such R genes are not resistant to all isolates of the pathogen, only those containing the specific avirulence gene recognized by P. infestans. Race-specific R genes often only provide short-lived resistance in the field as new virulent races of the pathogen rapidly overcome the resistance. A wild diploid potato species, Solanum bulbocastanum, is highly resistant to all races of P. infestans even under intense disease pressure (Helgeson et al. 1998). Somatic hybrids between cultivated potato and S. bulbocastanum were developed (Helgeson et al. 1998). Backcrossed progenies derived from the somatic hybrids showed durable and high level resistance in the field. A major resistance gene, RB, was mapped to chromosome 8 of S. bulbocastanum (Naess et al. 2000). The RB gene has recently been cloned and transgenic Katahdin (a highly susceptible variety) plants containing the RB gene showed broad-spectrum resistance against various strains of P. infestans in both greenhouse and field tests (Song et al. 2003). These results demonstrate that we should be able to develop potato varieties with high level of late blight resistance by introgressing the RB gene using a conventional breeding approach, such as marker-assisted selection.
A few of the somatic hybrids between S. bulbocastanum and potato are female fertile and have been used in the Wisconsin Potato Breeding Program. Backcrossed progenies between the somatic hybrids and elite breeding lines have been generated and used as parents. Each year approximately 5,000 seedlings are generated from these crosses. These seedlings have been pooled into the main breeding populations for selection. Large backcross three populations have also been created and are currently being evaluated for agronomic traits in order to be used as parents in further generations. Elite backcross three parents will be important for reducing the amount of linkage drag associated with the S. bulbocastanum genetic background. The major disadvantage of selection within the breeding lines created using the backcross material is not being able to select for disease resistance. We are unable to inoculate in the field, thus the only measure of late blight resistance is obtained during years of natural epidemics. Due to the large size of early breeding generations, it is also not possible to evaluate all lines.
A genetic marker has therefore been developed to easily track the presence of the RB allele in each individual potato and its subsequent progeny in the next generation. A 100% correlation was found between the presence of this marker and the late blight resistance phenotype. DNA is isolated from tuber shoots or plant leaves and a simple PCR reaction is performed. Hundreds of lines can be screened within one week, as apposed to the limiting field screens. Selection has been performed on populations known to be resistant (tests were performed with a detached leaf assay), as well as on more advanced field generations that have only undergone selection based upon agronomic performance (Colton et. al. 2006). It has been determined that the RB gene is transferred to the next generation at a rate of thirty percent, which is consistent with a monogenic allele being transferred in a tetraploid state.
First, this proposals aim is to continue selections upon the backcross three population, as well as the remaining S. bulbocastanum–related breeding lines that have been accumulated in our breeding program. We will analyze the ~500 single hills selected in the 2004 season. We will discard all of the clones that are negative for the marker. The future evaluation and selection of the marker-positive materials will be solely based on their agronomic performance. This will facilitate faster selection of environmentally friendly, RB-containing potatoes and help move these potatoes to the farmer in a more efficient manner.
Second, an initiative to inform the farmer about current molecular breeding methods will be undertaken. A field day with surveys will be conducted. Information and demonstrations will be used to illustrate the effectiveness of marker-assisted selection and the benefits of potatoes with the RB gene will be highlighted. Markers have been being developed for selections since the 1980’s, but few growers understand the value of this technique. Transgenics will be discussed and how these differ from marker-assisted selection, and how they are similar and are tracked using the same methods.
The short-term results that will be measurable by the end of this project will be a change in the potato grower’s knowledge regarding marker-assisted breeding. Growers will be informed of the marker-assisted selection process used to screen out only RB potatoes in the first generation of selection. A demonstration of how this narrows down the genetic base for the next generation will be performed. Somatic-hybrid development and subsequent breeding line development will be addressed. Growers will also become aware of the differences between somatic hybrids and transgenic or genetically modified potatoes.
Intermediate results will be a general understanding of how marker-assisted breeding benefits the grower. They will have an understanding that by using this form of breeding, cultivars with important characteristics will be available faster. Growers will have a better understanding of what type of resistance is offered by the RB gene and that less fungicide will need to be used.
Long-term outcomes from this project will be potato varieties containing RB-gene mediated late blight resistance.
Each of the female parents used to derive the nine breeding lines used in selection were derived from one of two original somatic hybrids between S. tuberosum and S. bulbocastanum, J101 or J103 (Figure 1). Each of these somatic hybrids was then backcrossed as the female parent to the popular S. tuberosum cultivar Katahdin. Katahdin is mainly a fresh market potato with good boiling and baking characteristics, but highly susceptible to late blight (Canadian Food Inspection agency, 2005). Line WTS 1218-4 was then backcrossed again to Atlantic, a standard chipping variety in the United States. All of the lines were then crossed to advanced lines from the Aberdeen Idaho Potato breeding program (A8495-1 and A86102-6), the Wisconsin Potato breeding program (W 1431 and W1355-1) or the North Dakota breeding program (ND(A) 2031). These crosses generated the backcrossed two (BC2) and one backcrossed three (BC3) female parents that were used to generate the nine lines whose progeny were tested for the presence of RB.
Six female parents, each carrying one resistant RB allele, were then backcrossed to advanced Wisconsin breeding lines (W 1099, W 11511, W 1836-3, W 1355-1 [Figure 2]). These will generate russet potatoes with superior frying and baking qualities with the benefit of late blight resistance. Crosses were also made with Stampede and Silverton russet potatoes which both have good fresh and frying characteristics. Dakota pearl, which has excellent chipping quality, was the final male parent used in the current line development. In total, 605 progeny were tested from the nine backcrosses, which should yield superior russet and chipping potatoes.
The crosses were made during the previous year at the Rhinelander Agricultural Research Station in Rhinelander Wisconsin. True seed was harvested during the winter of 2004 and planted during the spring of 2005. Germinated seedlings were transplanted into their own pots and allowed to mature over the summer of 2005. Multiple tubers were harvested from each of the individual plants. The tubers from each plant were separated into two identical flats. One set of flats was taken to the Department of Horticulture in Madison, Wisconsin for DNA isolation and PCR- marker-assisted selection. The remaining set of tubers was stored at 4 degrees Celsius at the Rhinelander Agricultural Research Station until planting the following spring.
DNA isolation and PCR
Potato tubers were stored at 4 degrees Celsius. Small pieces of tuber were cut out from the center of the tuber and placed into eppendorf tubes. Samples were crushed using a small pestle. DNA was isolated from tubers using a protocol similar to Williams, C. E. (1994), but with modifications for use with tuber sections (from A.H. del Rio, personal communication). Modifications included incubation of the tuber sample for no longer than 10 minutes at 65 degrees Celcius and precipitation of the DNA twice from the supernatant with isopropanol followed by an ethanol wash. PCR was then performed on all samples using the RB primers. Primer 1: 5′ C-ACGAGTGCCCTTTTCTGAC and Primer 1′: 5′ ACAATTGAATTTTTAGACTT. The 20-uL reactions were composed of approximately 50 ng DNA, 25 umol of each dNTP, 1 nmol of each primer, 0.2 units of Platinum Taq polymerase (Invitrogen, Carlsbad, CA) and 1x buffer with 15 mM MgCl2. The PCR was performed by the following protocol: 5 min at 95C; 35 cycles of 20 s at 95C, 20 s at 50C, and 1 min at 72C; followed by a final extension step of 7 min at 72C. PCR products were separated on 0.8% agarose gels in 1X Tris-borate EDTA and visualized and photographed with the Gel Doc 2000 (Biorad, Richmond, CA) under UV light after staining with ethidium bromide. DNA isolated previously and known to contain the RB gene was used as a positive control and DNA from a susceptible line was used as a negative control in all PCRs performed.
Grower Knowledge Assessment
In order to inform the potato growers in Wisconsin of the progress being made using marker-assisted selection multiple talks were given at field days and the major growers meeting in Wisconsin. Posters, presentations and visualization of GMO and conventionally breed potatoes were all used as tools to help the growers understand the different breeding techniques. Somatic hybrid generation was explained and compared to transgenic or genetically modified potato generation. Development of the marker used for selection was discussed in simple terms. The major outcomes of using this technology were discussed in detail. Specifically, how using MAS decreases the progeny from which field selections need to be made and the benefits of increased late blight resistance reducing input costs of fungicide, which in turn benefits the environment.
In order to assess the general knowledge of growers concerning the major topics I administered pre-surveys to the general audience at the WPVGA annual meeting. Post-surveys were taken after my talk at the WPVGA annual meeting and after the SpudPro meeting presentation. The pre and post- surveys were the same, but there were different versions which dealt with understanding concepts and trying to determine how growers feel about these concepts – if they are more likely to grow potatoes that have been developed using one method over another.
Potato Marker-Assisted Selection
The original parents for the nine lines tested were somatic hybrids between S. tuberosum (2n = 4x = 48) and S. bulbocastanum (2n = 2x = 24). Fusing one cell from each of these species, one would expect to obtain a new cell with all the chromosomes from each species (2n = 6x = 72). However, this is usually not the case. Previous work has been done which demonstrates a wide range of variability within the somatic hybrids themselves (Tek, A. personal communication). Current work supports this, finding somatic hybrid between S. tuberosum and S. bulbocastanum with varying degrees of wild and cultivated genome dosages. Polymorphisms were also detected that demonstrated rearrangements between the mitochondrial genomes (Iovene et. al. 2006). At the BC3 and BC4 level one would expect 98% of the genome to represent the recurrent parent, S. tuberosum. However, we cannot say this conclusively due to the variable nature of the somatic hybrid parents. It is also not known what types of chromosomal pairing will occur within these lines due to any rearrangements and unequal genome dosages. Therefore, we would estimate around 25 – 30 % of the next generation will contain a dominant allele in the simplex state.
In the original sample population, marker-assisted selection demonstrated that the transmission rate was around 30%. The current population sample is six times larger than the previous study, which may give us a better-unbiased proportion of RB alleles that are transmitted. Transmission of the gene using the current data is a little higher than expected, on average 45% of the progeny were positive for the RB gene. Within each line, one can see the variability of the transmission rate, which is also biased by the total number of progeny tested in each line (Figure 2). Of the parents that were repeatedly used in the generation of the nine lines, female parent WTS 1212-4 had a 46.5% transmission rate, male parent Stampede a 26% transmission rate and Dakota pearl was 51%, again demonstrating variability in transmission.
Grower Outreach / Survey Results
A total of 41 growers from around Wisconsin took the survey intending to gain a deeper understanding of their grasp of the scientific skills being used in breeding programs. This survey was intended to uncover the grower’s perceptions on these topics, not to actually assess the true knowledge they have about these topics. The survey was given at three different times, in two locations. The survey was administered to thirteen growers in the first general session meeting of the Wisconsin Potato and Vegetable Growers Association Annual (WPVGA) meeting in Stevens Point, Wisconsin to determine what the growers knew about these topics before they heard my presentations. The survey was then administered to sixteen growers after they had heard my presentation at the WPVGA meeting. Twelve growers also filled out the survey after hearing a presentation at one of the annual Spud-Pro meetings in Rhinelander Wisconsin. The survey was anonymous, so it is assumed that different growers took the survey each time.
The first portion of the survey asked the growers to rate their understanding of five main topics, specifically: somatic hybrids, marker-assisted selection, genetically modified organisms, resistance genes and marker-assisted breeding (Appendix I). It asked the growers to rate their understanding of these terms from never hearing of the concept to completely understanding the scientific applications and the mechanisms of the concept. In the general session, 61% of the growers had heard of somatic hybrids, but were unsure what they are or how they are applied. The remaining growers were split on somatic hybrids between never hearing of the concept to understanding how it is used in scientific study. In my session and the Spud-Pro meeting, after the growers had heard my presentation, they were still split on the concept of somatic hybrids. About 25% were found in each category, from hearing of the concept all the way through complete understanding of all scientific applications of somatic hybrids.
The next portion surveyed how well the growers understand marker-assisted selection. In the general session, growers were split between just hearing about this concept (38%) and understanding how this concept is applied scientifically (60%). In my session and the Spud-Pro meeting, growers increased their understanding of marker-assisted selection, but were still split in the degree of their understanding. Next, growers were asked how well they understand the concept of a genetically modified organism. In the general session, 75% said they understood this concept well, how it works and was developed, as well as its use in obtaining scientific results. The growers in my session were again split in their degree of understanding, but overall still 75% had the same understanding as the group from the general session. In the Spud-Pro meeting, 75% had a higher understanding of genetically modified organisms than the other two groups.
Growers were then asked about their understanding of resistance genes. Growers in the general session were split on their understanding of this topic, 31% understand how resistance genes work and are developed and 31% understand the mechanism of resistance genes and their scientific applications. In my session and the Spud-Pro meeting, growers had a high understanding of resistance genes (~75%). The final term growers were asked about their understanding of marker-assisted breeding. In the general session, 61% had just heard of marker-assisted breeding but were not sure what it is or how it is applied. In my session and the Spud-Pro meeting, the proportion of people who did not understand marker-assisted breeding decreased by 40% and most of those people moved up into a higher understanding of this concept.
The next question addressed how the growers feel about using genetically modified potatoes for breeding purposes. Their answers did not change depending upon what group was surveyed. On average, 50% had positive feelings, 15% negative and 28% neutral. Growers were then asked how they feel about using somatic hybrids for breeding purposes. In the general session 70% were neutral, while in my session and the Spud-Pro meeting ~60% were positive and ~40% neutral. The next questions varied between the different surveys given to the growers. This was done to try and get a wider variety of questions answered. In the general session and the Spud-Pro meeting, growers were asked if they have used cultivars derived from marker-assisted selection techniques. In the general session the grower were split between answering “no” or “I do not know.” The Spud-Pro growers were also split between “no” and “I do not know.” However, one grower did answer “yes.” Growers were also asked if they feel that marker-assisted selection is benefiting them. One grower said “no” in my session, while the other 88% either said “yes” or were “unsure”. In the Spud-Pro meeting 100% were “unsure” if marker-assisted selection was benefiting them.
The final two questions addressed how the growers felt about eating modified potatoes and about the RB gene specifically. Growers were asked if they would feel comfortable eating potatoes that had extra genes transferred via marker-assisted selection, genetic modification or only eating potatoes without extra genes – or they could also answer that they had no response. In the general session, most growers said they had no response or would only eat potatoes without extra genes. The growers in the Spud-Pro meeting were more positive about eating potatoes with genes from marker-assisted selection or genetic modification (70%). In my session, I asked the growers the same question, but asked about eating RB modified potatoes specifically. 78% were positive about eating potatoes with the RB gene transferred to potato using either method and 22% had no response. Finally, in my session and the Spud-Pro meeting, the growers were asked if they understand how the RB gene will be beneficial to potato growers in the future. 75-85% of the growers said “yes” and the remaining growers were unsure.
Educational & Outreach Activities
Field Days – with presentations and a poster:
All presentations were made out in the field along with the breeders of the Wisconsin Potato Breeding program (Bryan Bowen, Felix Nevaro and Jiwan Palta).
• Hancock Wisconsin – 8/9/2005
• Rhinelander Wisconsin – 7/14/2006
• Hancock Wisconsin – 7/26/06
• Stevens Point, Wisconsin – Wisconsin Potato and Vegetable Growers Association Annual Meeting – 2/15/2006: A 15-minute presentation directly to growers about MAS, different breeding strategies and GMOs.
• Rhinelander, Wisconsin – SpudPro meeting with the most active potato growers – 3/8/2006.
• Poster entitled “Continuation of Marker-Assisted Selection for Late Blight Resistant Potatoes” was presented at the Sixth International Solanaceae Conference Madison, Wisconsin – July 23 – 27, 2006.
• The information generated from this study will be documented in “Characterization of late blight resistant potatoes and the RB gene locus.” PhD thesis in Plant Breeding and Plant Genetics – expected completion date August 2008.
There is a higher overall transmission of the RB gene when looking at these lines. The breeding population has been significantly reduced using marker-assisted selection; 55% of the original population has been removed. This reduces the number of lines that will be screened in the field for favorable agronomic characteristics. The time needed to identify suitable parents for use in crosses has also dramatically decreased. Currently, those 275 potatoes that were selected from this study are being harvested and graded for specific agronomic qualities. Screening 275 potatoes will still give the breeders some flexibility. There is still quite a large amount of diversity remaining with which to select a parent containing superior qualities. By being able to continue the marker-assisted selection, the breeders can be confident that their selections will contain late blight resistance and use this to bring a superior potato to the growers of Wisconsin faster.
From the results of the grower surveys, the growers had a difficult time understanding the concept of a somatic-hybrid. Only about 30% of the growers understood the concept well after hearing my presentations. This was an improvement from the general session where more than half (61%) had only heard of the concept. I asked the growers about their understanding of marker-assisted selection and marker-assisted breeding. These two concepts are synonymous, and thus tested the growers understanding of breeding and selection. The number who answered in each column for these two concepts were approximately the same, indicating that most of the growers knew that these two concepts were the same. Growers appeared to perceive their understanding of genetically modified organisms as high. There were only a small percentage in each group of growers that answered the survey that had only heard of the concept and did not understand anything about genetically modified organisms. The growers also had a high understanding of resistance genes and it increased after hearing my presentations.
The survey then asked the growers about their feelings about genetically modified potatoes and somatic hybrids being used for breeding. It is interesting to note the growers feelings about using genetically modified organisms for breeding did not differ depending upon weather they understood the topic or not, nor what group they were in. However, growers did feel more positive about using somatic-hybrids for breeding after hearing my talk. It was interesting to find out that most growers did not know, or were sure they had never used a cultivar derived from marker-assisted selection. Most growers are also unsure if marker-assisted selection is even benefiting them at all. Many growers in Wisconsin are contracted with a specific company, such as Frito-Lay or a large grocery store chain, which determine which varieties they grow. Frito-Lay has a separate breeding program, which probably uses marker-assisted selection when possible. Growers are probably not aware they are using potatoes derived from somatic hybrids.
The last questions varied, but all dealt with what potatoes the growers would feel comfortable eating and how the RB gene will be beneficial. Growers that had heard my presentation were much more likely to eat potatoes with any genes or the RB gene specifically that had been transferred using marker-assisted selection or genetic modification. The growers that heard my talk also asserted that they do understand how the RB gene will benefit them in the future. There were still a few growers (~20%) that stated they did not know how obtaining cultivars with the RB gene would benefit them. Overall, most growers appeared to increase their knowledge about somatic-hybrids, genetically modified organisms and how the RB gene will be useful to them in the future after hearing my presentations.
An economic analysis was not performed at this time. Comparable yield, tuber shape, specific gravity and other agronomic characteristics (fry quality etc.) will be selected from the progeny of those potatoes containing the RB allele. In the future, once potatoes containing the RB allele are available to growers extensive field trial data will also be available on the reduction in fungicide use.
At this time, there has been no adoption of the potatoes developed in this study. Hopefully in the future, the growers will embrace a new potato with higher late blight resistance that was developed as a result of this study.
Areas needing additional study
It would have been beneficial to have more repeated parents in the study to determine if one parent had a greater chance passing along the RB allele. It would be useful in the future to perform selection on these same individuals for other genes that have been cloned recently and are known to add to late blight resistance or to give resistance to other pathogens. It also would be of interesting to do a more in-depth analysis on some of the plants to determine how the gene is being passed to the next generation. For example, performing cytogenetic analysis or looking at the genome region around where the gene is located in a few of the progeny would give us some insight.
More outreach to potato growers is needed on the difference between somatic hybrids and how they are different from genetically modified organisms. Most growers seemed to understand genetically modified organisms, but did not understand the distinction between a somatic hybrid. A larger sample size would have been ideal for obtaining a better representation of all growers. It also would have been ideal to actually test their direct knowledge of the main topics I covered and to measure if their stated understanding matched with their actual understanding of the topic. One area I did not address is if the growers even care about these forms of breeding and if it makes a difference in the type of cultivar they will grow. If they are in a contract with a company, do they really have a choice about the potatoes they grow? Are the growers concerned that they may be growing potatoes that have already been modified in some way?