Tag: plant breeding genomics

Objectives

After reading this learning module, you should be able:

• Define gene pyramiding/gene stacking
• Understand how molecular marker genotypic data can help guide gene pyramiding
• Calculate the population size required to obtain the desired gene combination based on parental genotype and location of genes within the genome

Introduction

Developing elite breeding lines and varieties often requires plant breeders to combine desirable traits from multiple parental lines, particularly in the case of disease resistance. The process of combining traits, known as gene pyramiding, can be accelerated by using molecular markers to identify and keep plants that contain the desired allele combination and discard those that don’t.

Selection based on molecular marker data (genotypic data) as opposed to traditional phenotypic data can confer many advantages. First, selection based on genotype allows breeders to identify and select desirable plants very early, such as the seedling stage, resulting in obvious savings of resources including greenhouse and/or field space, water, and fertilizer. Second, selection based on genotype can be cost effective when the cost of phenotyping is high and/or labor intensive. Third, when combining genes for resistance to the same disease, it can be difficult to distinguish, based on phenotype alone, those plants that carry all desired alleles from those that only have some of them. Fourth, unlike phenotypic selection, genotypic selection is not affected by environmental factors.

When using molecular markers to aid in the plant selection process, it is important to know where the molecular marker is located relative to the gene of interest. The genetic distance between marker and trait is calculated in genetic mapping studies (learn more about genetic mapping in an introduction to this topic provided by Wheat CAP). The farther a marker is from the DNA sequence polymorphism responsible for the trait, the greater the chance for recombination between the marker and the gene with each generation. If recombination occurs, selecting for a marker will not select for the trait, as the genetic linkage between the marker and the gene has been broken. This introduction to genetic mapping also outlines the process of genetic recombination.

• See an example of combining two disease resistance genes on the same chromosome into one line.

Minimum Population Size

When pyramiding genes, breeders must calculate the probability of an individual plant containing the desired combination of alleles. This probability dictates the population size required to have a high probability of finding at least one plant with the desired combination of alleles. Muller (1923) and Sedcole (1977) promote use of the following equation to determine the minimum population size required to recover an individual with the desired combination of traits:

N = log_e(1-P)/log_e(1-f)

where

• N is the minimum population size
• P is the desired probability of success (e.g. 99%, 95%, 90%)
• f is the frequency of the event (i.e., an individual plant having all desired alleles).

The frequency depends on the number of genes the breeder wants combined, whether the genes are genetically linked, and the breeding scheme being utilized. What follows are two examples of calculating f, as well as the minimum population size, for two scenarios: (1) combining two unlinked genes, and (2) combining two linked genes.

Combining two unlinked genes

In the simplest case, a breeder wishes to combine two unlinked favorable alleles from two different inbred parental lines into one variety. For example, we may want to combine Rx-3 and Rx-4, two genes that confer resistance to bacterial spot in tomato (Fig. 1). Rx-3 is located on tomato chromosome 5 and Rx-4 is located on chromosome 11. To combine the favorable alleles for these two genes, two inbred parental lines, one homozygous for Rx-3 and the other homozygous for Rx-4, would be crossed to generate heterozygous F₁ individuals. The F₁ individuals would be self-pollinated and F₂ individuals homozygous at Rx-3 and Rx-4 would be identified using molecular markers.

Figure 1. Bacterial spot on tomato fruit and leaves. Photo credit: David Francis, The Ohio State University.

Before the use of markers, breeders would need to grow progeny from F₂ individuals—F₃s—to differentiate between lines where a gene is homozygous dominant from those where the gene is heterozygous. If homozygous in the F₂, a gene will not segregate in the F₃, whereas if heterozygous, it will segregate. By allowing individuals with the desired genotype in the homozygous state to be identified after only two generations, DNA marker technology saves time and resources.

The question is: how large a population (number of F₂ individuals) must be grown to have a 99% chance of obtaining at least one individual that is homozygous at both gene loci?

To answer this, solve for N using Muller’s equation, N = log_e(1-P)/log_e(1-f).

In this case, P = 0.99

We can calculate f as follows:

The probability that an F₂ individual will be homozygous for one gene is 0.25. Therefore, the probability that an F₂ individual will be homozygous for two genes, is (0.25 x 0.25) = 0.0625. Thus, f = 0.0625. Learn about calculating expected genetic ratios).

Now we can calculate N by plugging P and f into Muller’s equation:

N = log_e(1-P)/log_e(1-f) = log_e(1-0.99)/log_e(1-0.0625) = 71.86.

Using the example above, a population with a minimum of 72 individuals must be grown to have a 99% probability of obtaining at least one F₂ individual homozygous for two unlinked genes.

This formula can be extended to estimate the minimum population size needed to obtain more than one individual with the desired combination of traits. Using the above example, to obtain 5 individuals homozygous for two genes, a population with 360 individuals (72 x 5) must be grown.

Combining two linked genes

What if a breeder wants to combine a gene from one inbred parent with a gene from another inbred parent, but the genes are linked? For example, we may want to combine Rx-3 and Pto, genes that confer resistance to bacterial spot and bacterial speck, respectively. Rx-3 and Pto are both located on tomato chromosome 5 (Yang et al., 2005). Suppose one inbred parental line is resistant to bacterial spot and the other is resistant to bacterial speck. To combine the resistance for bacterial spot and bacterial speck into one line, a recombination event must occur between the two genes, Rx-3 and Pto. The chance of such a recombination event is proportional to the distance between the genes. The closer the genes are, the lower the likelihood that a recombination event between the genes will occur. Consequently, more individuals must be evaluated in order to have a high probability of obtaining one with the desirable allele combination. In our bacterial spot and bacterial speck example, Rx-3 and Pto are located approximately 37 cM apart. To obtain one F₂ individual homozygous for the resistance alleles at both gene loci with a 99% probability of success, 133 individuals must be evaluated.

• Learn more about combining resistance to bacterial spot and bacterial speck.

Conclusion

Gene pyramiding is an important strategy for germplasm improvement. Pyramiding requires that breeders consider the minimium population size that must be evaluated to have a reasonable chance of obtaining the desired genotype. Molecular marker genotyping can facilitate the gene pyramiding process by reducing the number of generations that breeders must evaluate to ensure they have the desired gene combination.

References Cited

• Muller, H. J. 1923. A simple formula giving the number of individuals required for obtaining one of a given frequency. American Naturalist 57: 66–73. (Available online at: http://www.jstor.org/stable/2456535) (verified 29 Dec 2010).
• Sedcole, J. R. 1977. Number of plants necessary to recover a trait. Crop Science 17: 667–668.
• Yang, W. C., E. J. Sacks, M.L.L. Ivey, S. A. Miller, and D. M. Francis. 2005. Resistance in Lycopersicum esculentum intraspecific crosses to race T1 strains of Xanthomonas campestris pv. vesicatoria causing bacterial spot of tomato. Phytopathology 95: 519–527. (Available at: http://dx.doi.org/10.1094/PHYTO-95-0519) (verified 29 Dec 2010).

External Links

• Hain, P., and D. Lee. Backcross breeding 1 – basic gene inheritance [Online lesson] Plant and Soil Sciences eLibrary, University of Nebraska-Lincoln. Available at: http://plantandsoil.unl.edu/croptechnology2005/pages/index.jsp?what=topicsD&informationModuleId=957885794&topicOrder=1&max=7&min=0& (verified 5 Oct 2010).
• Shermin, J., and D. Quinn. Genetic mapping [Online lesson] Wheat CAP: Wheat Coordinated Agricultural Project. Available at: http://maswheat.ucdavis.edu/Education/animations/anim_mapping.htm (verified 7 Oct 2010).

Funding Statement

Development of this page was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.

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Photo credit: Patrick Hayes, BarleyWorld.org, Crop and Soil Science Department, Oregon State University

A variety of resources are available for those developing educational programs for barley producers. These include Fact Sheets developed by barley experts, presentations from previous barley workshops, podcasts, and videos.

• Fact Sheets
• Presentations
• Podcasts
• Videos

Fact Sheets

Barley: It’s What’s for Dinner. Fact sheet prepared by the Barley Coordinated Agricultural Project, written by Peggy G. Lemaux, in October 2007. This fact sheet discusses how barley is used as food, important preprocessing and food product traits of barley, and the forms of processed barley that are available. Funded by the USDA-National Institute of Food and Agriculture.

Barley, Malt and Beer. Fact sheet prepared by Barley Coordinated Agricultural Project, written by Karen Hertsgaard and Paul Schwarz, in June 2008. This fact sheet discusses how barley is used in malting, quality factors in malting barley, and an explanation of the malting and brewing process. Funded by the USDA-National Institute of Food and Agriculture.

Plant Breeding: The Path Forward. Fact sheet prepared by Barley Coordinated Agricultural Project, written by Peggy G. Lemaux, in July 2007. This fact sheet discusses the importance of plant breeding for barley. Funded by the USDA–National Institute of Food and Agriculture.

Marker-Assisted Selection. Fact sheet prepared by Barley CAP, written by Peggy G. Lemaux, in March 2008. This fact sheet explains marker-assisted selection, how it is used by breeders, and its benefits. Funded by the USDA–National Institute of Food and Agriculture.

Combating African Stem Rust in Barley. Fact sheet prepared by Barley CAP, written by Brian Steffenson and Peggy G. Lemaux, in October 2010. This fact sheet explains stem rust, its origins, potential impact on U.S. crops, and the steps being taken to prevent its spread. Funded by the USDA–National Institute of Food and Agriculture.

Barley Straw: A Potential Method of Algae Control in Ponds. Written by Bryan Butler, Dan Terlizzi, and Drew Ferrier in 2001 as part of Maryland Cooperative Extension’s Water Quality Workbook Series. This article addresses algae problems in Maryland, and the use of barley straw to control algae growth.

Algae Control with Barley Straw. This Ohio State University Extension Fact sheet, written by William E. Lynch, Jr., in 2002, discusses algae control with barley straw.

Presentations

Barley Varieties for South Central Montana, presentation by Ken Kephart, Geraldine Opena, and Tom Blake, Montana State University, in 2009. Presents yield data from variety trials, some dryland vs. irrigated.

Quality Matters!, a presentation by Paul Schwarz, North Dakota State University, in February 2009. Discusses the malting and brewing processes, malting quality parameters, impact of protein in malt, germination and sprouting, and Fusarium and gushing.

Barley Economics for 2009, a presentation by Dwight Aakre, North Dakota State University, in January 2009. Provides information on prices of inputs for malting barley and feed barley versus returns.

Crop Contract Considerations, a presentation by Frayne Olson, North Dakota State University, in January. 2009. Describes considerations that should be taken into account when negotiating a crop contract

Grain Storage, a presentation by Ken Hellevang, North Dakota State University Extension Service, in February 2010. Describes grain storage facilities, storage parameters, problems during storage, ways to ameliorate problems, aeration, polybag storage, and outdoor piles.

Estimating Crop Production Costs for 2010, a presentation by Dwight Aakre, North Dakota State University Extension Service, in February 2010. Describes production costs, including fertilizer, seed, fuel and pesticide costs.

Wheat and Barley Diseases 2010 – Best of Best Workshops, a presentation by Marcia McMullen, North Dakota State University Extension Service, in 2010. Includes information on wheat and barley pest surveys, early and late leaf disease control, fungicides, loose smut and viral diseases of barley.

Understanding Technical Signals in the Grain Futures Markets, an Idaho Barley Commission webinar presented November 10, 2010, by Craig Corbett, Idaho grain producer and market analyst. One-and-a-half-hour presentation. Uses charts and history to predict futures grain markets in same manner as oil futures and stock market predictions.

World Grain Market Outlook & 2011 Malt Contract Pricing, an Idaho Barley Commission webinar presented September 14, 2010, by Kelly Olson of the Idaho Barley Commission and Craig Corbett, Idaho grain producer and market analyst. One hour, 20-minute presentation (begins at 10-minute mark). Presentation includes a look at the 2010/2011 world grain market and supply and demand trends in barley, wheat and corn, and issues that can positively or negatively impact grain markets.

Podcasts

Combating the Threat of African Stem Rust. Podcast developed by Brian Steffenson, Peggy G. Lemaux and Barbara Alonso for Barley CAP, November 2010. This podcast explains stem rust, its origins, potential impact on U.S. crops, and the steps being taken to prevent its spread.

Videos

Perennial Grain Crops Could Be 20 Years Off. Video news highlight by WSU Today, featuring John Reganold, Washington State University, on movement to develop perennial versions of our major grain crops to address environmental limitation of annuals. Focused on wheat with a clear message for barley growers.

Funding Statement

Development of this page was supported in part by the National Institute of Food and Agriculture (NIFA) Barley Coordinated Agricultural Project, agreement 2009-85606-05701, administered by the University of Minnesota. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture. 

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Introduction

An important step in molecular breeding is the isolation of DNA, which allows plants to be genotyped or sequenced. DNA can be extracted on several scales, from one to hundreds of individuals at a time, and extraction can be done by hand or automated by using robots. For many procedures, only a small amount of leaf tissue—the size of a hole punch—is needed. Although specific protocols differ depending on the crop and extraction scale, the general steps to isolate DNA are as follows:

1. Plant tissue is harvested and placed in tubes or plates.
2. The tissue is homogenized to separate the cells from each other.
3. The plant cells and nuclei are lysed in the presence of an extraction buffer; the buffer contains salt and chemicals to help lyse the plant cells, stabilize the DNA, and reduce degradation.
4. The DNA is purified from cellular debris and other molecules such as proteins.
5. The DNA is precipitated by adding alcohol in the presence of salts.
6. The DNA precipitate is collected and washed.
7. The DNA is rehydrated in water or buffer solution.

Resources on DNA extraction

This video illustrates the basics of DNA extraction and gel electrophoresis in tomato. A transcript is available.

As part of their education and outreach, Wheat CAP developed a DNA extraction animation:

Photo credit: Wheat CAP.

External Links

• Sherman, J and D. Quinn. 2007. DNA extraction [Online animation]. Wheat CAP: Wheat Coordinated Agricultural Project. Available at: maswheat.ucdavis.edu/education/animations/anim_dna.htm (verified 11 May 2012).

Additional Resources

• Wikipedia contributors. DNA extraction. Wikipedia, The Free Encyclopedia. Available at: http://en.wikipedia.org/w/index.php?title=DNA_extraction&oldid=382504443 (verified 11 May 2012).

Funding Statement

Development of this lesson was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.

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SolCAP Workshops

2009 Potato Association of America SolCAP Workshop

Potato Association of America (PAA) 93rd Annual Meeting (August 9–13, 2009 Fredericton, New Brunswick, Canada)

SolCAP provided a workshop designed specifically for breeders in conjunction with the PAA. The presentations can be viewed by clicking on the topics below.

• SolCAP introduction
• Bioinformatics basics
• Marker technology
• Marker conversion
• Use of markers in potato breeding

2009 Tomato Breeders Roundtable SolCAP Workshop

The 43rd Tomato Breeders Round Table (June 28–July 1, 2009 Embassy Suites, Sacramento, CA)

SolCAP provided a workshop designed specifically for breeders in conjunction with the Tomato Breeders Round Table. The presentations can be viewed by clicking on the topics below.

• Marker technologies
• Analyzing quantitative trait loci
• Marker-assisted selection in tomato
• Effects of population structure on genetic analysis

SolCAP Workshop/Webinars

2010 Potato Association of America SolCAP Workshop/Webinar

The Potato Association of America 94th Annual Meeting (August 15–19, 2010 Oregon State University, Corvallis, Oregon)

• Working with the Potato Genome Webinar (Robin Buell)
• Working with Infinium Genotype Data Webinar (Allen Van Deynze)
• Linkage Analysis and QTL Mapping in Tetraploids Webinar (Christine Hackett)

2010 Tomato Disease Workshop SolCAP Workshop/Webinar

The 25th Annual Tomato Disease Workshop (November 16-18, 2010 University of Florida, Wimauma, Florida) 

SolCAP provided a workshop designed specifically for breeders in conjunction with the Tomato Disease Workshop. Presentation summaries, powerpoint slides, and webinars are available.

• Next Generation Sequencing (Allen Van Deynze)
• Accessing Available Sequencing Resources (David Francis)
• Introduction to the Tomato Genome Browser (Heather Merk)
• Bioinformatics 101 (David Francis)
• Working with Infinium Genotype Data (Allen Van Deynze)
• Downsteam Analysis with SNP Markers (Sung-Chur Sim)

About SolCAP

For more information about SolCAP (Solanaceae Coordinated Agricultural Project) please visit our web site at http://solcap.msu.edu (verified 16 May 2012).

Funding Statement

Development of this page was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.

Mention of specific companies is not intended for promotional purposes.

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In the first video clip, Dr. Allen Van Deynze, University of California, Davis, provides an overview of Sanger sequencing, next generation/second generation sequencing, and third generation sequencing technologies. In addition, the sequencing technologies are compared based on the amount of sequence generated, the cost, and the sequence read length.

If you experience problems viewing this video connect to our YouTube channel or see the YouTube troubleshooting guide.

In the second video clip, Dr. Van Deynze provides visual representations of the portion of the genome sequenced using different sequencing technologies, and he discusses the potential utility of the sequence, from expressed sequence tags (ESTs), to the transcriptome, to the whole genome.

If you experience problems viewing this video connect to our YouTube channel or see the YouTube troubleshooting guide.

Find all the presentations from the 2010 Tomato Disease Workshop

Additional Resources

For a review of sequencing technologies,

• Thompson, J. F., and P. M. Milos. 2011. The properties and applications of single molecule sequencing. Genome Biology 12: 217. (Available online at: genomebiology.com/2011/12/2/217) (verified 25 Feb 2011).

Funding Statement

Development of this lesson was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.

Mention of specific companies is not intended for promotional purposes.

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Figure 1. Bacterial Spot of tomato. (a) Bacterial spot symptoms on tomato fruit. (b) Defoliation due to bacterial spot. Photo credits: David Francis, The Ohio State University.

Introduction

Bacterial spot causes yield loss in tomato through reduced photosynthetic capacity and defoliation. Fruit quality is reduced indirectly due to sun-scald and directly by bacterial lesions. Bacterial spot of tomato is caused by as many as four species of Xanthomonas, including Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In addition, at least five physiological races—T1–T5—are recognized on the basis of a hypersensitive (HR) reaction on a differential series of host genotypes. Descriptions of bacterial spot may use the species names, taxonomic groups A–D, or race designations.

References

• Jones, J. B., G. H. Lacy, H. Bouzar, G. V. Minsavage, R. E. Stall, and N. W. Schaad. 2005. Bacterial Spot – Worldwide distribution, importance and review. Acta Horticulturae (ISHS) 695: 27–34. (Available online at: http://www.actahort.org/books/695/695_1.htm) (verified 01 Mar 2012).

Additional Resources

• Miller, S. A., R. C. Rowe, and R. M. Riedel. 1996. Bacterial spot, speck, and canker of tomatoes. Factsheet HYG-3120-96. The Ohio State University Extension. Available online at: http://ohioline.osu.edu/hyg-fact/3000/3120.html (verified 01 Mar 2012).
  
• Momol, T., J. Jones, S. Olson, A. Obradovic, B. Balogh, and P. King. 2002. Integrated management of bacterial spot on tomato in Florida. Publication #PP192. University of Florida IFAS Extension. Available online at: http://edis.ifas.ufl.edu/pp110 (verified 01 Mar 2012).
• Sun, X., M. C. Nielsen, and J. W. Miller. 2002. Bacterial spot of tomato and pepper. Plant Pathology Circular No. 129 (revised) [Online]. Florida Department of Agriculture & Conservation Services. (Available at: http://www.doacs.state.fl.us/pi/enpp/pathology/pathcirc/pp129rev.pdf) (verified 01 Mar 2012).
• Asian Vegetable Research and Development Center Bacterial Spot (Xanthomonas campestris pv. vesicatoria) [Online]. AVRDC International Cooperators’ Fact Sheet. (Available at: http://www.avrdc.org/LC/tomato/bactspot.html) (verified 01 Mar 2012).

Funding Statement

Development of this lesson was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.

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