Overview

This lesson explains the importance of the backcross method in the development of improved crop varieties. The theory behind the method, plus its strengths, weaknesses and requirements will be discussed.

Authors

• P. Stephen Baenziger, Department of Agronomy and Horticulture, University of Nebraska-Lincoln
• Patricia Hain, Department of Agronomy and Horticulture, University of Nebraska-Lincoln

Published 2023

Updates completed by P. Stephen Baenziger in 2023 with McKinzie Sutter.

Introduction to Backcross Breeding

With all of the advances in molecular biology, it may seem surprising to find out that traditional plant breeding methods are still needed in the development of plant varieties. Today, the backcross procedure is most often used to move a transgene from a good tissue culture variety that was used in transformation to an elite experimental line or variety. It turns out that for many crops, once the transgene is in the crop species crossing is more efficient than transformation procedures. Backcrossing is more efficient than transforming the elite line because most transformation protocols are optimized for a specific (often poorly adapted and lower yielding) laboratory line. Many elite lines (which are high yielding) are not amenable for transformation. Hence genetic engineers transform their lab line and breeders backcross the transgene from the lab line into the elite line.

In this lesson the focus will be on the backcross method, which is a form of recurrent hybridization (repeated crossing to a single variety; the recurrent parent) where a superior characteristic may be added to an otherwise desirable variety or elite line. In this method the breeder has considerable control of the genetic variation in the segregating population in which selections are to be made. The backcross method has been used extensively for transferring qualitative characters (characters with clear phenotypes that are easy to identify in cross progeny) such as disease resistance. It is effective in both self and cross pollinated crop species. To better understand the applications of backcrossing, the gene for leaf rust resistance in wheat will be used as an example. Figure 1 shows the visible symptoms of leaf rust in susceptible wheat. The left picture leaves are resistant as indicated by a small amount of rust. The plant on the right leaves are covered with rust, indicating it is susceptible.

Figure 2 illustrates how the backcross procedure can be used to move leaf rust resistance (RR, Rr; note the dominant gene determines the gene symbol, in this case capital R) from one variety to a susceptible variety (rr).

The actual procedure for back crossing is almost self-explanatory. In back crossing you have a donor parent (has a gene of interest) and a recurrent parent (an elite line that could be made better by adding the gene of interest). The donor parent is crossed to the recurrent parent which is used as the female parent to ensure any heterozygous Rr progeny are the result of a cross and not a self. The progeny of this cross is then crossed to the recurrent parent (it is 'crossed back' to the recurrent parent, hence the term back cross). The progeny of this cross is selected for the trait of interest and then crossed back to the recurrent parent. This process normally is repeated for as many back crosses as are needed to create a line that is the recurrent parent with the gene of interest from the donor parent. The goal of backcrossing is to obtain a line as identical as possible to the recurrent parent with the addition of the gene of interest that has been added through breeding.

In the end, you want to keep only the individuals homozygous for the resistance gene. To obtain them, self heterozygous Rr plants from [latex]BC_4[/latex]. The resulting offspring will be 1RR : 2Rr : 1rr. Progeny testing would be needed to identify homozygous dominant RR from heterozygous Rr plants. Progeny testing is where the genotype of a parent plant is determined by genotypes of the line’s progeny and often their phenotypic expression. In the case of an RR plant, the progeny will all be RR (no segregation for the gene/trait). However in the case of a heterozygous Rr plant, the progeny will segregate [latex]\frac{1}{4}[/latex] RR : [latex]\frac{1}{2}[/latex] Rr : [latex]\frac{1}{4}[/latex] rr. Therefore, the progeny of RR plants will be uniformly resistant to leaf rust, while the progeny of Rr plants will segregate for resistance and susceptibility.

In contrast, if the genes for rust resistance had been recessive (i.e., ss = resistant) rather than dominant (SS; again the dominant trait determines the gene symbol, in this case capital S), then the introduced resistant gene is only carried in the heterozygote and would not be detected throughout the backcross program. After each backcross, one would have to self the heterozygote (Ss) in order to produce resistant plants (ss) in the progeny. These resistant plants (ss) are then backcrossed to the recurrent parent (SS). See Figures 3a and 3b. Note in this case, the SS parent is used as the female parent to ensure any heterozygous Ss progeny was the result of the cross and not a self.

When working with recessive traits, such as this example, Allard (1960) suggests advancing the 1st backcross to the [latex]F_2[/latex] generation followed by selection for the desirable character from the donor parent (ss) and the general features of the recurrent parent. The 2nd and 3rd backcrosses are then made in succession after which the inbreeding with selection phase for ss is repeated. This is followed by the 4th and 5th backcrosses in succession. The [latex]BC_5F_2[/latex] that are resistant (homozygous ss) are crossed to recurrent parent (homozygous dominant SS) for the [latex]BC_6F_1[/latex] which is heterozygous Ss.

The above backcrossing protocols are using phenotypic selection for the trait of interest. With the advent of molecular markers, backcrossing can be facilitated by selecting for a molecular marker closely linked to the gene of interest. The molecular marker is usually dominant or codominant, hence the protocol in Figure 2 is used in marker assisted backcrossing.

Genetic Basis for the Backcross Method

In the [latex]F_1[/latex], many loci will be heterozygous (those loci which had different gene alleles coming from the two parents, Fig. 4).

These loci will include the gene of interest as well as many other loci containing genes for other traits. With backcrossing, homozygosity for the alleles from the recurrent parent will increase at the same rate as is seen with the approach to homozygosity with selfing (one form of inbreeding). The formula for calculating this rate is given below:

[latex]{\LARGE \left(\frac{2^n - 1}{2^n}\right)^m}[/latex]

where n = number of backcross generations and m = number of loci

Example of homozygosity from selfing:

Heterozygous Rr is backcrossed to homozygous recessive rr.

Rr x rr [latex](2^1 - 1)^1 \text{ or } \LARGE \frac{(2 \cdot 1) - 1}{2^1} = \LARGE \frac{1}{2}[/latex] homozygosity

[latex]BC_1[/latex]: [latex]\frac{1}{2}[/latex] Rr : [latex]\frac{1}{2}[/latex] rr

In the [latex]BC_1[/latex] generation, half the plants are homozygous for the recurrent parent allele and half the plants are heterozygous for the recurrent and donor parent allele. Similarly, in the [latex]F_2[/latex] generation, [latex]\frac{1}{2}[/latex] the plants are also homozygous (RR or rr, note they are not homozygous for only one of the two alleles).

Let’s think back to our original example of leaf rust resistance (RR, Rr). One of the goals of backcrossing is to remove the donor parent’s genes (except the one of interest, RR; the others are nontarget genes) and increase the recurrent parent’s genes except for the gene of disinterest (rr). The amount of remaining genetic information (the nontarget genes), on the average, from the nonrecurrent parent (donor parent) is reduced by 50% with each backcross (Fig. 5).

The calculation for this data is:

(RR, Rr) Percentage of nontarget genes from donor parent = [latex]\left(\frac{1}{2}\right)^{n+1}[/latex]

where n = number of backcrosses.

However, the rate at which genes entering a cross from the donor parent (R = rust resistant) are eliminated during backcrossing will be influenced by linkage. Linkage is a term used to describe genes that do not independently assort (one of the requirements for Mendelian inheritance). Linked genes tend to segregate (stay) together and unlinked genes independently assort. The physical basis for linkage is how close the genes are to each other on a chromosome and how frequently crossovers occur between the genes. If genes are far apart or on different chromosomes, they are unlinked and independently assort. If they are near to each other on the chromosome they are more frequently passed on together than separately (Fig. 6).

Linkage is measured by the recombination frequencies/map distance. Factors such as centromere location and chromosome structural abnormalities can reduce crossover events during meiosis. For example, if an undesirable allele d, for dwarfing is linked to R (rust resistant), and selection is only for R, d tends to be brought along in the [latex]F_1[/latex]. However, when reintroducing R each backcross, the number of opportunities for crossing over between the R and d loci increases. Therefore, the probability of eliminating d is:

[latex]1 - (1-p)^n[/latex]

where, n = number of backcrosses and p = recombination frequency between loci.

It should be noted that if d and R are close together (small map distance), it will be very hard to select R and eliminate d. In this case linkage reduces breeding progress and increases the number of backcrosses needed to eliminate d. However, it should be remembered that linked molecular markers to the gene of interest are needed for marker assisted backcrossing. In this case linkage between the marker and the gene is very beneficial. In practice, rarely do breeders know all the traits which are linked to the gene of interest in the donor parent selected for a backcrossing program.

General Considerations

When starting a backcrossing program, there are a few items that a breeder needs to consider.

First of all, backcrossing is most easily conducted if the character being added is easily selectable. To be easily selected it needs to be: simply inherited (although the backcross approach may be applied to quantitative traits, especially if molecular markers are used to select the gene of interest); dominant; and easily recognized in the hybrid plant. An example of an easily selected trait is awns (recessive trait) on wheat heads (Fig. 7) and a less easily selected trait is dough strength (affected by the environment and generally under the control of many different genes; Fig. 8). See the Cereal Quality video below for some examples of grain qualities (no audio on the video).

There are also some absolute requirements for a successful backcross program.

First is a satisfactory recurrent parent. Good candidates for the recurrent parents are those varieties or inbred lines that have maintained their importance for many years despite the release of numerous ’improved’ varieties intended to replace them. A recurrent parent that has maintained its importance is one that remains high yielding in comparison to other lines over time. Remember that it often takes 5 to 7 years to develop a backcross line. In that 5 to 7 years, new cultivars are developed. If the new cultivars with higher yield are better than the recurrent parent with the new trait, no one will grow the recurrent parent with the new trait. Thus the time and effort to back cross the new trait into the recurrent parent would be wasted except as a parent to develop other new cultivars.

Secondly required is a useful gene that is unaffected by environment or being in a new genetic background. High heritability of the character being transferred is important for phenotypic selection. Transfer is easiest when the character can be identified readily in hybrid populations by visual selection, by simple tests, or by linked molecular markers. There has been some success in dealing with quantitative traits through backcrossing; however, like all other methods it depends on the ability of the breeder to distinguish between genetic and environmental variability, and to select those individuals that are desirable for genetic reasons. The general agricultural worth of the donor parent need not be of great concern. Selection of the donor, (nonrecurrent parent) is almost exclusively on the basis that it exhibits the character in a particularly intense form. This high gene intensity is important since often the presence of modifier genes in the new genetic background (recurrent parent) cause some intensity to be lost even though the most stringent selection has been practiced throughout the backcrossing program.

A final absolute requirement is a sufficient number of backcrosses must be used to reconstitute the recurrent parent to a high degree if that is the goal. Recovery of the recurrent parent is a function of the number of backcrosses and the effectiveness of selection for recurrent parent type in the early generations. Usually six backcrosses with selection for type in the early generations has proved sufficient. However, in wide crosses and/or with undesirable linkages a greater number of backcrosses may be necessary. Remember that in practice the objective is to improve the recurrent parent by only one trait. It should be noted that since the recurrent parent is already a proven variety or line, it is usually not necessary to conduct extensive performance trials once satisfactory introduction of the desired character has been achieved. Previous data on the recurrent parent will indicate the benefits of the backcross line assuming the added gene or linked genes are not detrimental.

The last consideration a breeder is aware of is the influence of environmental conditions on a backcross program. Provided the expression of the character being transferred is sufficient for selection, backcrossing can be conducted in any environment. For example, several generations may be grown per year in the greenhouse (Fig. 9). Again, tightly linked molecular markers that are not affected by the environment to the gene of interest can be extremely useful in ensuring the backcross procedure can be done in any environment.

Improvement of More Than One Character

Often in a variety development program, the breeder wants to add more than one gene to a variety. This is known as gene stacking. It is possible to introduce several different characters in the course of a backcross program (Fig. 10). Usually the other characters being added have already been introduced into the recurrent parent in other backcross programs. For example, one breeder wanted to develop rust and bunt (another disease) resistant Baart 38 wheat. He did this by backcrossing rust resistance into Baart and by backcrossing bunt resistance into a second line of Baart. He then crossed the rust resistant Baart and the bunt resistant Baart and selected Baart that was both rust and bunt resistant.

Currently, backcrossing is often used to develop improved gene pools or selection populations without trying to return completely to the recurrent parent’s phenotype. In some cases, the variety has a good phenotype and the breeder wants to improve it without having a clearly defined trait (for example, yield).

In order to meet this objective, the breeder can use different numbers of backcrosses (1 to 6) to add as much or as little of the recurrent parent as he/she/they think is necessary. A backcross one population (A x (A x B)) is preferred by many breeders to a simple three-way cross (A x (B x C)), which may have too much variation. In this case A, B, and C represent elite lines or varieties.

Strengths and Weaknesses

There are both strengths and weaknesses of the backcross breeding method. Properly executed, the backcross breeding methods allow all the desirable characteristics of the recurrent parent to be recovered with the possible exception for characters governed by genes tightly linked to the gene(s) being transferred. This may be considered a strength. The backcross method provides a certain and precise way of making predictable gains with little possibility that uncontrolled segregation will produce subtle weaknesses in the variety.

This same characteristic may also be considered a weakness. The method sets an upper limit which is equal to the recurrent parent’s phenotype. This limit will often be lower than the progress that is possible with other lines when segregation is not rigidly controlled. This phenomenon is known as yield lag and is graphically depicted in Figure 11. Series 1 shows the gradual percent improvements reached over time for a given breeding program that is active for 6 years. In contrast, Series 2 is put through a backcross program at year 4, where its percent improvement plateaus.

Conclusions

The backcrossing method remains an efficient tool for transferring one or a few genes into established crop varieties. With each succeeding backcross generation, a greater proportion of the recurrent non-target genes remain along with the donor's gene of interest. There are several considerations and other absolute requirements to take into account when developing a backcross program to ensure a successful end result.

Acknowledgements

The development of this lesson in 2000 was supported in part by Cooperative State Research, Education, & Extension Service, U.S. Dept of Agriculture under Agreement Number 98-EATP-1-0403 administered by Cornell University and the American Distance Education Consortium (ADEC). 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 U.S. Department of Agriculture.

References

Acquaah, G. (2012). Principles of Plant Genetics and Breeding (2nd ed.). Wiley-Blackwell.

Allard, R. W. (1960). Principles of plant breeding. John Willey and Sons. Inc. New York, 485. ISBN: 0 471 02310 8. See Chapter 14.

Fehr, W. (1991). Principles of cultivar development: Theory and technique. ISBN: 0-9635989-0-2. See Chapter 28.