Homozygosity – True-Breeding Lines, Their Route and Their Proof

Homozygosity means true breeding: at a given place in the genome the plant carries the same variant twice. Such places are called loci. A plant that is homozygous at the loci in question passes the corresponding traits on unchanged, and a line of such plants is called a pure line or an inbred line.

The most useful thing to know about it is that you cannot see it on the plant. The quantity of cannabinoids formed, meaning the active compounds breeders sort by, depends heavily on growing conditions; only the ratio of the two main ones to each other follows the genome. Appearance and genetics come apart precisely at the traits used for selection. Without a laboratory measurement or a tested progeny, any claim about a true-breeding line is a guess.

What it is needed for

For cross breeding, true breeding is the condition under which two particular parents always yield the same progeny. Maize shows the pattern: its inbred lines differ from one another, yet each is true-breeding in itself, which is why the cross between two particular lines turns out the same every time. Only that makes a variety reproducible rather than a one-off.

How far “stable” sits from true-breeding

A variety that has looked the same for years need not carry anything identical at its loci. The classic chemotype study worked with lines that had already been inbred twice, and even so, depending on the combination, between 5.6 and 37.5 % of the places scored segregated in the first daughter generation – in nearly a third of the combinations, more than a third of them. A remainder of mixed loci on that scale is enough to make a progeny inconsistent, and it was invisible on the plants.

The two routes there

Two routes lead there, and they differ in pace. One is sib-mating, the pairing of two plants from the same progeny; in dioecious species, meaning those carrying male and female flowers on separate plants, it is the route the breeding literature works with, and it requires both sexes to be present. The other is selfing, the fertilisation of a plant with its own pollen. In cannabis that is only possible through induced sex reversion, in which a female plant lays down male flowers.

Selfing is the faster of the two, and that pace carries a cost the time calculation leaves out. At equal levels of inbreeding, sib-mating clears out the variants that are lethal in double copy more effectively: in the model, 7.11 such variants remained under sib-mating against 9.3 under selfing, because homozygosity rises step by step rather than in one jump. The figure comes from a model of natural populations; what transfers is the mechanism, not the number. On top of that, every round of selfing needs the sex reversion and therefore selects for the capacity to perform it – the finished line carries a raised risk of unwanted male flowers, however carefully the work is done.

For selfing, the stretch has been measured in this species. Under single seed descent, in which one individual plant is carried forward per generation, it typically takes six generations for the share of mixed loci to fall below 3 %, starting from 17 to 35 % in open-pollinated source plants; the measurement ran against a panel of 1,500 markers, markers being places in the genome that can be read unambiguously in the laboratory. Approaches via doubled germ cells have so far failed in this species. True breeding is therefore a threshold in practice rather than an absolute state.

What the stretch costs

In the line whose seven generations were grown side by side, the share of abnormal flowers rose from 0.4 % in the source generation to 13.7 % in the sixth, more than thirtyfold: hermaphrodite flowers, flowers in places where none belong, and intermediate forms of the male and female floral parts.

From the fourth selfing generation onward, meaning after four consecutive rounds, the plants set seed only when different parts of the same plant were treated at once with one agent that releases the ripening gas ethylene and a second that blocks its effect. That is something other than inbreeding depression, the plain loss of vigour and yield: here the inbreeding reaches into the control of sex expression itself. Without that double intervention on a single plant, the line runs out at this point.

That close mating costs vigour is not peculiar to this species. In maize, vigour usually drops below half that of open-pollinated varieties during inbreeding and returns only in the cross between two unrelated inbred lines.

How true breeding is demonstrated

Two routes remain, and both take more than a look at the plant. In the laboratory the measurement runs over a marker panel with the 3 % threshold named above. Without a laboratory, progeny testing remains: a line is constant only once its offspring are. Either way it costs a generation or a lab, which is exactly why so many lines circulate as “stable” that never were.