18) How to do “Backcrossing” clone dubbing

" How “Cubing the Clone” works

A)
In this first situation, we will address the case where a plant breeder discovers a special individual or clone.

It is natural to be curious and to cross a few plants that attract us. Grow them and find a new variation that you like even more. We can preserve this new variation through cloning indefinitely, but accidents can happen, and the clones can die. They can catch viruses or suffer from clonal depredation due to somatic mutations over time. Additionally, it is more challenging to share clones with friends by mail than seeds. Therefore, it is entirely reasonable to want to create seed backups of this special clone.

But before we start breeding this clone, we need to try to determine exactly what we expect from the seeds we are going to create. Do you simply want them to reproduce individuals like the special clone? A simple backcross (cubing) will accomplish that. Or do you want to create seeds that can produce more seeds like the special clone, a truly breeding strain? These are very different things. You see, there is a strong chance that your special clone is heterozygous for many traits it expresses phenotypically. This simply means it will contain genetic information (genes) for two opposing traits, but you can only see one, the dominant one. However, its seeds will only receive one or the other of the genes, so its offspring will express all the genetic information it possesses, including what you cannot see in itself. If you want to create a truly breeding strain, you need to preserve all the genes you can see and eliminate all the genes you cannot see but that may appear in the offspring. Create homozygosity. The only way to achieve this is through selection and generational inbreeding (selecting homozygous offspring to be the parents of the next generation).

Backcrossing and Cubing

Backcrossing is when you cross an individual (your special clone) with its offspring. It's weird in our world, but plants seem to like it.

• Your first backcross is simply a backcross.

• Your second backcross, where you take the offspring from the first backcross and cross them again with the SAME parent (grandparent now), is often called SQUARING by plant breeders.

• Your third backcross, where you take the offspring (square) from the second backcross and cross it again with the SAME parent (now the great-grandparent), is often referred to as CUBING by plant breeders. You can continue the backcrossing, but it's simply called backcrossing after that. Cubing refers to the number three, as in 3 backcrosses.

Cubing operates on the basis of mathematical probabilities in relation to genetic frequencies. The more males you use for each crossing, the more likely your reality will match the theory. In theory, with the first backcross, 75% of your genetic pool will match the genetic pool of the P1 parent during cubing. Squaring increases this to 87.5%, and cubing raises it to 93.75%. You can arrive at these figures by taking the average between the two parents that make up the cross. For example, you start by crossing the mother P1 (100%) with an unrelated male (0%), resulting in (100% + 0%) divided by 2 = 50%. Therefore, the offspring from this first cross is loosely considered to be 50% like the mother. Take them and make your first backcross, and you get (100% mother + 50%) divided by 2 = 75%. And that’s how we arrive at the 75% for the first backcross. The same logic applies as you perform more backcrosses. As you will see later, you can apply this same probability mathematics to specific genes or traits, which can have a dramatic effect on your methodology and selection methods.

Your selection of the right males for each backcross is the critical point for successfully employing this technique. In each case, you could select males carrying the genes you want, or you could accidentally choose individuals harboring unwanted recessive genes. More likely, you might simply select individuals that are heterozygous for both genes, just like the mother P1 during backcrossing. The simplest way to manage this is to start by focusing on one gene and one trait. For instance, let’s assume that flavor is determined by a single gene (which is probably not the case in reality). You can create a few Punnett squares to demonstrate genetic frequencies across three generations of backcrossing. Now, suppose we have found an individual with a special pineapple flavor in our population of pine-flavored plants that we want to preserve. The gene causing the pineapple flavor could be dominant or recessive, and the selection capabilities and outcomes of cubing will differ in each case.

P = pineapple flavor et p = pine flavor

Therefore, since each individual will have two paired flavor genes, the possible genotypes are PP, Pp, and pp. Since P is dominant, both PP and Pp will express the pineapple flavor, while pp will exhibit the pine flavor; these are their phenotypes. Now, since pineapple is a new flavor, it is likely that the special individual is heterozygous, or more specifically, Pp. Consequently, the only possible parental combination is Pp × pp, with Pp being the parent to be cubed.

Figure 1 The F1 cross:

Most people will struggle to select males carrying the pineapple flavor gene, as males do not produce female flowers. Therefore, they will randomly and blindly select males based on this trait. The ratio of the P and p genes in the male F1 generation to be used in the first backcross will be 2:6. Another way to look at it is to say that the frequency of the P gene is 25%. This means that one out of every four pollen grains will contain the pineapple flavor gene. Here’s how it unfolds in the first backcross.

Figure 2: The B1 Cross:

"This first backcross creates, in the first place, a homozygous individual (PP) for the pineapple flavor. However, once again due to our limited selection abilities, we choose the males at random. Among the randomly selected males, we should expect that three out of the eight pollen grains will contain the pineapple flavor gene. The female P1 will always contribute a P gene for each p gene. I will spare your computer's memory and not publish the table; feel free to do it yourself on paper to ensure you understand what is happening."

The second backcross (Squaring) will produce the following:

3 PP 8 Pp 5 pp

Therefore, 68.75% will have a pineapple flavor and 31.25% will have a pine flavor. The P gene frequency increased to 7/16, or 43.75%.

And finally, the third backcross (Cubing) will produce the following genotypic ratios:

7PP 16Pp 9pp

Therefore, 71.875% will have pineapple flavor after the cubing is completed. About 22% (7/32*100) of the cubing offspring will be pure breeding for pineapple flavor. The P gene frequency has increased to about 47% (30/64).

In conclusion, if backcrossing continued indefinitely with random selection of males and with a large enough population size, the P gene frequency would plateau at 50%. This means that the best one could hope for from cubing is 25% of pure breeding for pineapple flavor and 75% that will display pineapple flavor. You will never get rid of the 25% that will retain the pine flavor. This pattern would hold true when trying to cube any heterozygous trait.

B)
Pineapple taste is recessive In this case, P corresponds to pine taste and p to pineapple taste.

"The convention is that the uppercase letter signifies dominance. For the breeder to have noticed the interesting trait, the mother to be cubed should be homozygous for the pineapple flavor (pp). Depending on the origin of the male and whether it is related, it could be Pp or PP, with PP being more likely. This will not make much difference in the outcome. The F1 cross is quite basic, so we will skip the diagram. We simply cross the female (pp) with the male (PP) and obtain offspring that will all be Pp. Since the pine flavor is recessive, none of the F1 descendants will have the pineapple flavor (hint). However, the frequency of the p gene will be 50%. pp X PP = Pp + Pp + Pp + Pp Since the F1 generation is all the same (Pp), the pollen it produces for the first backcross will contain a p gene for each P gene. The first backcross will be: B1 = pp X Pp = Pp + Pp + pp + pp As you can see, 50% of the offspring will have the pineapple flavor, and the frequency of the p gene is 6/8, or 75%. This B1 generation will produce pollen containing 6 p genes for every 2 P genes."

Figure 3 The second backcross:

"As you can see, the second cross or square produces a pineapple flavor in 75% of the offspring. And the frequency of the p gene in this progeny is about 88% (Remember C88). Among the pollen grains from this cross, 14 out of 16 will carry the p gene for the pineapple flavor. When they are backcrossed with the mother P1 for the third time, they produce the following cubed offspring."

Figure 4 The third backcross:

After cubing a pair of homozygous genes, about 88% of them will exhibit the desired trait (the pineapple flavor in this case) and will also be purebred for that same trait. The frequency of this desired gene will be approximately 94%. If the backcrossing were to continue indefinitely, the frequency of the gene would continue to approach 100%, but would never completely reach that goal.

It is important to note that the above examples assume no selection pressure and population sizes sufficient to ensure random matings. The smaller the number of males used in each generation, the greater the selection pressure, whether intentional or not. The importance of the breeding population size and selection pressure is much greater when the traits being cubed are heterozygous. Moreover, the examples above only consider a single pair of genes.

In reality, most traits that we select for similar potency are influenced by multiple characters. The calculations then become more complicated if you want to determine the success rate of a cubing project. Generally, you multiply the probabilities of obtaining each trait in relation to one another. For example, if your pineapple trait were influenced by 2 distinct recessive genes, you would multiply 87.5% * 87.5% (0.875 * 0.875 * 100) and get 76.6%. This means that 76.6% of the offspring would have the pineapple flavor. Now suppose the pineapple trait is influenced by 2 recessive traits and one heterozygous dominant trait. We would multiply 87.5% by 87.5% by 71.9% (0.875 * 0.875 * 0.719 * 100) and get 55%. By simply increasing to three genes, we have reduced the number of cubed offspring with a pineapple aroma to 55%. Therefore, cubing is a good technique when you want to increase the frequency of a few genes (this is an important point to remember), but as the project grows, the chances of success decrease... at least without a certain level of selective pressure.

Apply pressure

The best way to significantly increase your chances of success is to apply intentional selective pressure and eliminate unintentional selective pressure. Try to find clear and effective ways to isolate and select for and against certain traits. Find ways to ensure that your males transmit the intended traits and eliminate all males that do not. This includes ALL traits that can be selected. Some traits will be directly observable in males, while others, such as flowering time, may not be. If you are selecting for a trait that cannot be directly observed, you need to perform progeny testing to determine which males are passing on the most desirable genes. I will explain progeny testing in more detail later.

It is important that when you select your best males, you ignore superficial traits that have nothing to do with the true traits you are seeking. You see, cannabis has several thousand genes residing on only 10 pairs of chromosomes or 20 individual chromosomes. Therefore, each chromosome contains hundreds of genes. It is said that each gene residing on the same chromosome is linked to another. Generally, they travel in groups. If you select one of them, you are actually selecting all the traits on that chromosome. There is an exception to this rule called the breaking of linked genes by crossover, but for simplicity, we will ignore that for now. Returning to selection, you might decide to select a trait like you like the pointed appearance of the leaves while being truly interested in fixing the grapefruit flavor. But it could happen that the two traits are on the same pair of chromosomes but on opposite chromosomes. If that is the case, as long as you select the plants with pointed leaves, you will never achieve the grapefruit flavor that you really desire. It is good to keep in mind that each time you select a trait, you are selecting several hundred genes. This is why most serious breeders learn to take small, methodical steps and work on one or two traits at a time, especially with inbreeding projects such as self-fertilization and backcrossing.

Now let's see what kind of improvements we can make to the first example of trying to cube a heterozygous dominant trait using some selective pressure. Let’s say that with each generation, we are able to eliminate the recessive individuals for the pine flavor (pp), but we cannot eliminate the heterozygotes (Pp). If you remember, our mother P1 had the genotype (Pp) in this model, and the F1 cross produced (Pp + Pp + pp + pp) as possible combinations of offspring. We eliminate the two individuals (pp), leaving us with only Pp. Therefore, our first backcross will be:

Pp * Pp = PP + Pp + Pp + pp

We again eliminate the individual pp, leaving us with PP + 2Pp. Entering the second backcross, we have increased our frequency of the P gene from 37.5% to 66.7%. This means that entering the second backcross, 4 out of 6 pollen grains will carry the P gene. The result is as follows

Figure 5:

As you can see, after selecting against the homozygous recessives for 2 backcrosses, we have increased our frequency of the P gene from 44% to 58% in our squared population. If we eliminate the homozygous recessives again, our gene frequency increases to 70% (14/20) in the third backcross, meaning that 7 out of 10 pollen grains will carry the P gene. Again, I will save your PC's memory and simply provide you with the results of the third backcross.

Crossing B3 = 7 PP + 10 Pp + 3 pp

This means that 95% of the offspring will have the pineapple flavor after cubing a heterozygous dominant strain if the homozygous individuals with the pine flavor are eliminated before each backcross. This is an improvement over the 72% when no selection took place. The frequency of pure breeding individuals for the pineapple flavor has risen to 35%. But more importantly, the frequency of the P gene improves to 60%. This will be an important factor to consider when we discuss progeny testing.

But for now, let’s summarize the percentage of breeding individuals for the pineapple flavor in each of the models. In the case where the pineapple flavor trait is heterozygous dominant and no selective pressure is applied, cubing produced 22% breeding individuals. By selecting against the homozygous recessive pine gene, we were able to increase that figure to 35%. And finally, by cubing a homozygous recessive gene, we are able to obtain a cubed population that is 87.5% breeding for the pineapple flavor. And as I pointed out earlier, these figures apply only to traits governed by a single gene. Suppose the pineapple flavor is coded by two distinct genes, one dominant and the other recessive, and you can select against the homozygous recessive gene for the pine flavor while selecting for the dominant gene for the pineapple flavor. Your cubed population would then contain 87.5% * 35% (0.875 * 0.35 * 100) = 30% breeding individuals. As you can see, as long as the cubing source is heterozygous, no matter how many backcrosses you perform, you will never achieve a true breeding strain.