RESULTS
Variation in cannabinoid composition:
Examples of Cannabis gas-chromatographic profiles are shown in Fig 1A. The variation found within accessions is usually at both levels of cannabinoid type (different retention times) and amount (different peak areas), confirming other authors' observations ( S MALL and B ECKSTEAD 1973; DE M EIJER et al. 1992 ). Plants with only a single dominant cannabinoid (THC or CBD in this study) are present as 95-98% of the total cannabinoid content ( Fig 1A, top and middle chromatograms). Such plants are here termed as pure chemotypes and can be found naturally occurring within accessions or can be produced by crossing or self-fertilizing plants showing a mixed chemotype ( Fig 1A, bottom chromatogram; see below).
S 2 inbred lines:
If the clone originally used to produce the S 2 was of a pure CBD or THC chemotype, this chemotype is preserved throughout all the subsequent inbred generations, although the absolute amount of the dominant cannabinoid still shows considerable variation, as demonstrated by the standard deviations found ( Table 1 ).
The molecular analysis performed on the S 2 lines suggested a narrowing of the genetic variation within these materials, especially if compared with noninbred populations as examined previously ( F ORAPANI et al. 2001 ). The percentage of polymorphisms detectable by RAPD analysis (three primers) ranged from 17.3 to 46.4% ( Table 1 ), while it was previously reported to be from 65 to 80% within the most common Italian and French fiber cultivars ( F ORAPANI et al. 2001 ).
F 1 hybrids:
When the S 2 plants were mutually crossed as indicated in Table 2, all F 1 plants examined contained both CBD and THC in considerable amounts, and no pure chemotypes were found. An example of the distribution of the F 1 plants (F 1 99.3) in a CBD vs. THC plot is presented in Fig 2, where the values for the parental plants are also indicated. Fig 2 shows a heterotic effect for the total cannabinoid content (CBD + THC) and in particular a strong increase of the CBD content of the F 1 's as compared with the parental CBD content. Similar patterns were found in two of the other F 1 's. However, there was no such effect on the total cannabinoid content in the 99.4 F 1.
Figure 2. CBD vs. THC content for the 99.3 F 1 progeny. The positions of the F 1 plants are indicated by open triangles; their parents (belonging to the CBD line 94.4.12.63 and to the THC line 55.22.7.10, respectively) are indicated by open squares. The cannabinoid content is expressed as weight percentage of the dry, mature inflorescence.
Surprisingly, the CBD/THC ratio appeared to vary in a clear progeny-specific way. The average ratio varied significantly ( P < 0.001) from 0.50 (F 1 99.4) up to 1.57 (F 1 99.5), where only four plants could be analyzed ( Table 3 ).
Table 3. CBD/THC ratios of heterozygous plants
The molecular analysis of the F 1 plants was again carried out by using three RAPD primers and comparing the loci identified in the parental plants with those present in the F 1 progenies. In the four progenies, from 5.6% (99.3) to 37.5% (99.4) of the RAPD loci scored in the parental plants segregated in the F 1 (data not shown). This indicates that a still significant number of loci were in the heterozygous state in the S 2 parentals.
F 2 inbreds:
The CBD vs. THC contents of one of the F 2 's examined are plotted in Fig 3. Within each F 2, the individuals could unmistakably be assigned to three different segregant groups on the basis of large discontinuities in the calculated CBD/THC ratios. Data on the segregation of chemotypes in the different F 2 's are shown in Table 4. For all F 2 's, the results of the 2 test accepted the model of a single locus with two codominant alleles. In those F 2 's where the segregation ratios deviated most from 1:2:1 (largest 2 values), this was consistently due to an underrepresentation of pure CBD chemotypes.
Figure 3. CBD vs. THC content plot of the 99.6.14 F 2 progeny (circles). The positions of the single F 1 plant (open triangle), self-fertilized to obtain this F 2, and of the initial pure-CBD and pure-THC parents (open squares) are indicated. Cannabinoid content is expressed as weight percentage of the dry, mature inflorescences.
Table 4. Chemotype segregation data
As in the F 1 's, the CBD/THC ratio for heterozygous plants in the F 2 's again appeared to be strongly progeny dependent. ANOVA and a multiple-comparison least-squared difference (LSD) test performed on heterozygous CBD/THC ratios of all evaluated F 1 and F 2 progenies showed highly significant differences among progenies ( P < 0.001; Table 3 ). Especially, the 99.4 progenies form a very distinct cluster as they are composed of heterozygotes having a much higher proportion of THC than of CBD. In all other progenies the proportion of CBD in heterozygotes exceeds, to a varying extent, the THC proportion. Small, though significant, differences in mean CBD/THC ratio do occur among progenies sharing the same pedigree. However, the F 1 CBD/THC ratios (assessed in a different year from the F 2 ratios) are transmitted with very little change to the heterozygotes of the corresponding inbred F 2 's.
