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Advanced Backcross Breeding

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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 trangene 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) where a superior characteristic may be added to an otherwise desirable variety. 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, they have a small amount of rust. The plant on the right is covered with rust, it is susceptible.

The figure shows two examples of wheat, the one on the left is mostly green with very little rust showing, so it is resistant. The reaction type is type 1 (small pustule surrounded by a chlorotic halo). The wheat on the right is covered with orange rust spots so it is susceptible. The reaction type is type 4 (Large pustules with no chlorosis or secrosis).

Fig. 1. Two examples of wheat, the one on the left is resistant to rust,
the one on the right is susceptible to rust.

Donor parent is Rust resistant variety (Big R Big R--resistant and dominant) and the Recurrent parent is Adapted variety 'A' (little r little r--susceptible) The picture shows the result of crossing a Donor parent with a Recurrent parent and then at each generation crossing a Recurrent parent with the result of the previous generation.  At the first generation, the F1 progeny are all big R little r. The next generation is called BC1 progeny (backcross 1) and is a cross of F1 and the Recurrent parent little r, little r.  This is done for multiple generations, each time using a big R little r progeny and crossing it with the Recurrent Parent.  The progeny will segregate big R little r (resistant) : little r little r (susceptible) so you inoculate the progeny plants and discard the susceptible progeny and cross to the resistant progeny.
Fig. 2. Backcross breeding with a dominant trait.

Figure 2 illustrates how the backcross procedure can be used to move leaf rust resistance (RR, Rr) 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. 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 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.

                      (The above animation is in Flash and may not be viewable on some devices.)
 

In the end, you want to keep only the individuals homozygous for the resistance gene. To obtain them, self Rr plants from BC4. The resulting offspring will be 1RR : 2Rr : 1rr. Progeny testing would be needed to identify RR from Rr plants. Progeny testing is where the genotype of a parent plant is determined by genotypes of the line’s progeny. In the case of an RR plant, the progeny will all be RR (no segregation for the gene/trait). However in the case of an Rr plant, the progeny will segregate 1/4 RR : 1/2 Rr : 1/4 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.

When the trait you want to add is controlled by recessive genes, a different approach is needed.  The F1 is big S little s (susceptible), so you need to self the F1 to get the F2 in the ratio one big S big S to two big S little s (susceptible) to one little s little s (resistant).  If you inoculate the selfed progeny, you discard the susceptible plants and cross to the resistant plants.  You repeat this process in every back cross generation (cross and then self, select the resistant selfed plants for crossing in the next back cross).  This is a diagram of Backcrossing with trait = recessive: Donor parent as Rust resistant variety (little s little s --resistant and recessive) and the Recurrent parent as Adapted variety 'A' (big S big S --susceptible and dominant) It shows the result of crossing a Donor parent with a Recurrent parent to produce the F1 generation. Since the desired trait is recessive, the progeny will all be big S little s, so the F1s must be selfed to produce a homozygote little s little s. At each generation, the recessive homozygote must be crossed with the recurrent parent and the resulting progeny must be selfed to once again get a recessive heterozygote which is then crossed with the recurrent parent.
Fig. 3. Backcross breeding with a recessive trait.

In contrast, if the genes for rust resistance had been recessive (i.e., ss = resistant) rather than dominant, 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 Figure 3.

When working with recessive traits, such as this example, Allard (1960) suggests advancing the 1st backcross to the F2 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 BC5F2 that are resistant (SS) are crossed to recurrent parent (SS) for the BC6F1 which is Ss.  The BC7F1 is selfed to get in the BC6F2 : 1/2 SS (susceptible) : 1/2 Ss (susceptible) : 1/2 ss (resistant) backcross with intense selection for both the desired character (ss) and the recurrent parent plant phenotype. You have successfully transfered the gene.(If interested, see Allard, p. 156-157, for further description and rationale for this approach.)

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