Inbred line
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An inbred line is a line of plants bred by mating close relatives for many generations, usually by self-fertilisation or by mating full siblings, until its plants are homozygous at nearly every locus.[1] Each generation of selfing halves the heterozygosity that remains in a line. Lines started from one population become fixed for different alleles, while the frequency of each allele over all the lines does not change.[2] A cross between two inbred lines gives an F1 generation whose plants are alike and heterozygous at every locus where the parent lines differ. Such crosses often show heterosis.[3][4]
Cannabis is pollinated by the wind, is mostly dioecious and naturally outcrosses. Even a single cultivar is genetically variable.[4] Inbred lines of cannabis are made by inducing flowers of the other sex on a plant so that it can pollinate itself, by self-pollinating monoecious hemp under pollen-proof isolators and by crossing siblings by hand.[1][5][4] In Russia, self-pollinated lines of monoecious hemp have been selected for trace THC content and stable monoecy and used as parents of crosses.[5] Inbred lines are the parents of F1 hybrid seed, whose production in cannabis is still limited.[1][4] Inbred lines and their hybrids have also been used to assemble cannabis genomes and to breed mapping populations.[6][7] Some lines sold as seed under the label IBL, for inbred line, are kept by open pollination within the line.[8]
From a population to inbred lines
When a plant heterozygous at a locus, Aa, is selfed, a quarter of its offspring are AA, a quarter aa and half Aa. A plant that is homozygous gives only homozygous offspring, so in a line kept by selfing the heterozygosity left after t generations is (1/2)t of what the line began with. The inbreeding coefficient, the probability that a plant's two gene copies at a locus are identical by descent, rises towards one.[2] A Russian programme of self-pollinated hemp lines planned on the same rule: the share of heterozygotes at one pair of alleles was to fall from 100 per cent in the starting plants to 50, 25, 12.5, 6.25, 3.125 and 1.5625 per cent over six generations.[5]
Lines started from one population become fixed for different alleles. In Felsenstein's example, the population carries two alleles at frequencies of 0.6 and 0.4. After seven generations of selfing, 59.8 per cent of the lines are AA, 39.8 per cent aa and 0.4 per cent still Aa. The frequency of A over all the lines does not change, although within any one line it goes to zero or one.[2] Crossing different lines therefore restores much of the heterozygosity of the base population. Keeping a population as isolated inbred lines would hold on to more of its alleles than any system of mating within one population, but the system is rarely used in domestic animals and plants because of the effects of inbreeding on viability and fertility.[9] Those effects, the expression of deleterious recessive alleles and the loss of heterozygotes at loci where they are fittest, are inbreeding depression.[4]
A dioecious line can be inbred by mating siblings, but it becomes homozygous more slowly than under selfing.[4] After the first few generations a line kept by mating full siblings loses nearly 20 per cent of its remaining heterozygosity each generation.[10]
These expectations assume that there is no selection. Where a trait goes with vigour or fitness, natural selection during inbreeding is likely to hold the mean of a line above the expected value, and selection at linked loci slows the course of inbreeding depression.[3] Measured heterozygosity does not fall by the same amount every generation. In cannabis lines inbred by single-seed descent, heterozygosity at 1,500 SNPs began between 0.17 and 0.35 and fell by amounts that differed widely from one generation to the next. Six lines came below 3 per cent, the level the authors took as functional homozygosity.[4] The k-mer counts of the genomes of the highly inbred cultivars CBDRx, FL18 and FL49 show signs of residual heterozygosity.[6]
Making inbred lines in cannabis
A female cannabis plant can be made to form male flowers with silver thiosulfate, which inhibits the plant hormone ethylene, and a male plant to form female flowers with ethephon, which releases it. The induced flowers are fertile, so a plant of either sex can be self-pollinated.[4] A selfed female gives only female offspring, since its own pollen carries only X chromosomes.[1] Monoecious hemp bears male and female flowers on one plant and can be selfed without treatment.[4] The use of selfing to produce feminised seed, and the depression that follows it in cannabis, are described under outcrossing.
Self-pollinated lines of hemp
At the Penza Research Institute of Agriculture, V.A. Serkov and L.V. Klimova self-pollinated plants of six low-THC monoecious hemp cultivars and 11 of the institute's own breeding selections in 2006, to breed hemp of the Central Russian type. They grew the lines from 2007 to 2011. Each plant was enclosed, as its flower buds began to form, in its own isolator, 70 by 30 centimetres, of a light non-woven fabric that let moisture through but kept out foreign pollen. The isolator was adjusted as the plant grew and taken off after flowering.[5]
In the first two generations, low field germination of the selfed seed, which the authors put down to inbreeding depression, limited how many lines could be grown. Between 17 and 72 lines were grown in each of the first five generations. From the second generation some lines had trace THC contents, below 0.01 per cent, while others had more than 0.2 per cent. Some lines produced no ordinary male plants among their monoecious ones, and one did so in all five generations. Eight lines of the sixth generation were chosen as parents for crosses. Between them they combined trace THC content below 0.009 per cent, no ordinary males, oil contents above 32 per cent and total fibre yields above 32 per cent.[5] Serkov and Klimova list what such a line needs as a parent: a high content of fibre or oil, good general and specific combining ability, a high seed yield, little cannabinoid content, stable monoecy and little inbreeding depression.[5]
Inbreeding of hemp had been studied in the Soviet Union before them. I.A. Sizov, one of the first to study it, found that inbreeding weakened the plants and proposed it for fixing monoecy. Later studies found depression in the first selfed generation in 25 to 30 per cent of lines. Lines came to differ in height, branching, panicle length, leaf form and colour and the length of the growing season, and some threw dwarf plants with pale green or yellow stems and leaves.[5] The recessive characters that appeared in these lines are described under dominance and recessivity.
Single-seed descent
In single-seed descent a line is carried forward from one plant in each generation. García-de Heer and colleagues wrote in 2026 that single-seed descent was the most practical way to make homozygous parent lines in cannabis, though a slow one, needing about six generations to approach functional homozygosity.[4] They inbred 16 accessions from the genebank of the Leibniz Institute of Plant Genetics and Crop Plant Research in Germany and from a private collection in Australia. In each generation the most vigorous seedling of the chosen sex was isolated in a pollen-proof chamber. Plants of dioecious lines were treated on one branch with silver thiosulfate or ethephon. Up to six rounds were completed in 18 months, and five lines reached the sixth selfed generation.[4]
From the fourth generation onwards every line produced abnormal flowers and set less seed. In one line whose seven generations were grown side by side, abnormal flowers rose from 0.4 per cent in the starting plants to 13.7 per cent in the sixth selfed generation. Treating different parts of a plant with both silver thiosulfate and ethephon restored seed set. Selfing through female plants of dioecious lines failed most often, in 8 of 22 attempts, because the male flowers induced on them in later generations shed little pollen. Monoecious female lines failed in 4 of 27 attempts and lines inbred through XY plants in 2 of 12. The authors advised inbreeding dioecious cannabis through male plants where possible. Selfed XY plants gave male and female offspring in about equal numbers, with no sign of viable YY plants.[4] Two of the inbred lines had a lower total dry weight than the accessions they came from and two did not differ. The authors suggested that choosing the most vigorous seedling in each generation may account for part of this.[4]
Doubled haploids
In other crops a line that is completely homozygous can be made in a single generation from a haploid plant of male origin whose chromosomes are doubled, spontaneously or by treatment.[11] In 2021 a week of cold treatment of excised male flower buds turned a very small share of their microspores towards embryo development, the first induction of androgenesis in cannabis. Only a few structures with several nuclei formed, and none developed further.[11] García-de Heer and colleagues wrote in 2026 that methods of inducing doubled haploids had so far been unsuccessful in cannabis.[4] Serkov and Klimova note that spontaneous haploids can occur in up to 3 per cent of hemp plants.[5]
Inbred lines as hybrid parents
Seed of a cross between two inbred lines gives plants that are genetically alike. Seed of outcrossed cannabis gives variable plants, which has led growers of medical cannabis to propagate their crops as clones.[4] In the account of Barcaccia and colleagues, the drug cultivar Skunk No. 1, bred in the early 1970s, was made by taking plants of an F2 progeny through nine cycles of inbreeding to raise their homozygosity. Ten females and ten males were then propagated as clones and crossed in all pairwise combinations. The authors call the method effective for breeding highly heterozygous synthetic varieties, especially when the parents are tested for their general combining ability.[1]
Barcaccia and colleagues write that true F1 hybrid varieties were not bred in the past because inbred lines pure enough to be used every year as parents were hard to keep. Only recently have some seed companies produced them, keeping the male and female parent lines as clones. Where those parent clones are not fully homozygous, the hybrid progeny segregates and its plants vary.[1] Heterosis often comes from each parent masking the deleterious recessive alleles of the other, so inbred lines are tested for their specific combining ability in crosses in all pairwise combinations, a diallel design. Two, three or four inbred lines can be combined into two-way, three-way or four-way hybrids. Markers are used to check the homozygosity and genetic stability of each line.[1]
The single-seed-descent lines of García-de Heer and colleagues were crossed in five F1 combinations. The hybrids were more uniform than either the inbred lines or the original accessions, and all but one were larger than their inbred parents. Four of the five set more seed than their inbred parents or the original lines. Only two had every plant within the range of 20 per cent either side of each stated constituent that Australia's Therapeutic Goods Administration requires of dried flower products, for every cannabinoid measured. The hybrids outside that range came from parents of different chemotypes.[4] In the Russian programme, the self-pollinated hemp lines of the sixth generation went into crosses in 2012.[5]
Inbred lines in genome research
Besides breeding, homozygous lines are used in quantitative trait locus analysis, mutagenesis, gene isolation and genome mapping.[4] At a locus where two inbred lines are fixed for different alleles, every F1 plant is heterozygous, and the F2 segregates one-quarter AA, one-half Aa and one-quarter aa.[3] De Meijer and colleagues crossed inbred cannabis plants with pure CBD and pure THC chemotypes to study how chemotype is inherited.[12] The parents of the mapping population used to assemble the CBDRx genome, the drug cultivar Skunk No. 1 and the fibre hemp Carmen, had each been crossed between siblings for five generations to raise their homozygosity. CBDRx, which the NCBI chose as its reference genome for cannabis under the name cs10, is itself highly inbred.[6]
The cannabis pangenome published in 2025 includes EH23, an F1 of two proprietary female inbred lines: ERB, a high-CBDA line that is day-neutral, and HO40, a high-THC line that is sensitive to day length. Each of its two haplotype-resolved assemblies stands for one parent. EH23 was also selfed, after male flowers had been induced with silver thiosulfate, to make an F2 mapping population.[7] Long stretches of homozygous sequence in an assembly can show how a line was bred. The assembly of FL 58, which Stack and colleagues call an inbred line made by several generations of self-pollination, has runs of homozygosity along almost the whole length of chromosomes 2, 3, 4, 7 and 8 and the X chromosome.[13]
The term in the seed trade
Some seed producers sell lines labelled IBL. The Hemp Depot listing of ATAO Genetics' Mazar-I-Shariff IBL F5 describes a line begun in 2009 from a female selected from landrace seed and kept as a clone. It was reproduced twice outdoors, to an F2 and a backcross, and its third to fifth generations were raised indoors by open pollination.[8] A line kept by open pollination among several plants inbreeds more slowly than a line kept by selfing. In an idealised population of N plants, a fraction 1/(2N) of the heterozygosity is lost each generation through genetic drift.[14]
See also
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Barcaccia, G.; Palumbo, F.; Scariolo, F.; Vannozzi, A.; Borin, M.; Bona, S. (2020). "Potentials and challenges of genomics for breeding cannabis cultivars". Frontiers in Plant Science. 11 573299. doi:10.3389/fpls.2020.573299.
- ↑ 2.0 2.1 2.2 Felsenstein, J. (December 2019). Theoretical evolutionary genetics. Seattle: Department of Genome Sciences, University of Washington. pp. 231–233.
- ↑ 3.0 3.1 3.2 Felsenstein, J. (December 2019). Theoretical evolutionary genetics. Seattle: Department of Genome Sciences, University of Washington. pp. 411–414.
- ↑ 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 4.15 4.16 Garcia-de Heer, L.; Mieog, J.; Burn, A.; Nolan, M.; Liu, L.; et al. (2026). "Weeding out variability: a proof-of-concept for producing uniform F1 hybrid Cannabis sativa L. using single-seed descent". Horticulture Research. 13 (5) uhag038. doi:10.1093/hr/uhag038. PMC 13188225.
- ↑ 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 Serkov, V.A.; Klimova, L.V. (2016). "Sozdanie i izuchenie samoopylennykh linii dlya prakticheskoi selektsii nenarkoticheskoi konopli posevnoi" [Creation and study of self-pollinated lines for the practical breeding of non-narcotic seed hemp]. Niva Povolzh'ya (in русский) (2 (39)): 52–60.
- ↑ 6.0 6.1 6.2 Grassa, C.J.; Weiblen, G.D.; Wenger, J.P.; Dabney, C.; et al. (2021). "A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana". New Phytologist. 230 (4): 1665–1679. doi:10.1111/nph.17243. PMC 8248131.
- ↑ 7.0 7.1 Lynch, R.C.; Padgitt-Cobb, L.K.; Garfinkel, A.R.; Knaus, B.J.; Hartwick, N.T.; et al. (2025). "Domesticated cannabinoid synthases amid a wild mosaic cannabis pangenome". Nature. 643 (8073): 1001–1010. doi:10.1038/s41586-025-09065-0. PMC 12286863.
- ↑ 8.0 8.1 "Atao Seeds". Hemp Depot. Retrieved 8 October 2026.
- ↑ Felsenstein, J. (December 2019). Theoretical evolutionary genetics. Seattle: Department of Genome Sciences, University of Washington. pp. 242–244.
- ↑ Felsenstein, J. (December 2019). Theoretical evolutionary genetics. Seattle: Department of Genome Sciences, University of Washington. p. 237.
- ↑ de Meijer, E.P.M.; Bagatta, M.; Carboni, A.; Crucitti, P.; Moliterni, V.M.C.; Ranalli, P.; Mandolino, G. (2003). "The inheritance of chemical phenotype in Cannabis sativa L.". Genetics. 163 (1): 335–346. doi:10.1093/genetics/163.1.335.
- ↑ Stack, G.M.; Quade, M.A.; Wilkerson, D.G.; Monserrate, L.A.; Bentz, P.C.; et al. (2025). "Comparison of recombination rate, reference bias, and unique pangenomic haplotypes in Cannabis sativa using seven de novo genome assemblies". International Journal of Molecular Sciences. 26 (3) 1165. doi:10.3390/ijms26031165. PMC 11818205.
- ↑ Felsenstein, J. (December 2019). Theoretical evolutionary genetics. Seattle: Department of Genome Sciences, University of Washington. pp. 254–256.