ANDA SUJETO DE LA MANO PUNTO CLAVE DEL

Twenty different fragment profiles were produced across 26 samples examined in the initial primer-trial using the snoR29F/snoR30 primer combination. Fragment sizes ranged from 205 to 260 bp in length and were therefore shorter than the size recorded in A.

thaliana(284 bp) (Figure 4.3). The number of fragments per sample varied between one

(e.g.S. madagascariensis(22)) and six (e.g.S. massaicus(6)).

Fragment profiles varied between species and different fragment patterns were found between samples within species apart from within S. flavus (Figure 4.3) A high level of fragment pattern variation was evident within S. glaucus where all samples differed in number and sizes of their profiles and only one fragment (216 bp) was shared betweenS. glaucussamples (5) and (17) (Figure 4.3).

A number of fragments were shared between hybrids and their parent species (Figure 4.3). While S. squalidus (sample 3) shared only one band (227 bp) with one

Chapter 4 Results parent, S. aethnensis (sample 1), three fragments (216, 220 and 227 bp) of S. squalidus

(sample 10) were shared with its parents (S. aethnensis (1): 216 and 227bp and S.

chrysanthemifolius (2): 220bp). However, three of the S. squalidus(10) fragments (216,

220 and 250) were also present inS. vulgaris (7) and (8), the latter sharing three bands (216, 220 and 243 bp) with its hybrid S. teneriffae (11). S. cambrensis (9) shared two fragments (216 and 250 bp) with each of its parents, S. squalidus (10) and S. vulgaris. The bands seen in S. flavus (at 216, 226 and 260 bp) were also present in its hybrid S.

mohavensis(19, 20, 21) (Figure 4.3). Due to low number of samples examined for each

species the detection of hybrids based on shared fragments between hybrid species and one of its parents is of limited value. However, the results obtained here might be useful for choosing regions for a more detailed investigation.

205 208 212 213 214 216 220 221 222 224 226 227 240 243 248 249 250 260 1 S. aethnensis 2 S. chrysanthemifolius 3 S. squalidus 10 S. squalidus 7 S. vulgaris

8 S. vulgaris var.hib.

9 S. cambrensis 11 S. teneriffae 12 S. teneriffae 5 S. glaucus 16 S. glaucus 17 S. glaucus 4 S. massaicus 6 S. massaicus 13 S. flavus 14 S. flavus 15 S. flavus 19 S. mohawensis ssp.bre. 20 S. mohawensis 21 S. mohawensis 22 S. madagascariensis 23 S. madagascariensis 24 S. madagascariensis 25 S. madagascariensis 26 S. madagascariensis 27 S. madagascariensis

Samples Species snoR29/snoR30 fragment sizes (bp)

Figure 4.3: Fragment profiles of Senecio ssp. generated using primers snoR29F/snoR30R. Twenty different fragment profiles were generated among 26 samples surveyed across 13 species/subspecies. The matrix shows fragments obtained for each sample.hib.=hibernicus;brev.=breviflorus.

Chapter 4 Results In the NJ tree, some samples clustered according to species. For example, all S.

madagascariensis samples, except S. madagascariensis (26), were placed in the same

cluster.S. madagascariensis(26) shared one of its two fragments withS. mohavensisand

S. flavus and was placed within a cluster containing these species. However, for some

species, different samples were placed in different clusters within the tree. For instance, samples of S. squalidus,S. teneriffae andS. glaucuswere present in two different clades at the base of the tree. The species within these two clades are intermixed and no clear hybrid-parent relationship is seen. Furthermore, S. cambrensis is not found within these clades, but is placed at the base of the clade containing most of theS. madagascariensis

samples. However, the hybrid speciesS. mohavensisis placed withS. flavus, which is one of its parents.

Chapter 4 Results

Figure 4.4: NJ tree of Senecio sp. based on fragment variation generated by snoR29F/snoR30R primers.(hib.=hibernicus;brev.=breviflorus).

Chapter 4 Results

4.3.1.2 Summary of all clusters

Most primer pairs that successfully amplified products in the initial primer-screen revealed variation between and within species and species groups (i.e. the S. squalidus

group plus S. vulgaris and S. teneriffae) (Table 4.1). However, the amount of variation between and within species depended on the cluster/primer pair examined. For example,

S. flavus, S. mohavensis and S. madagascariensis often possessed distinct fragment

profiles and formed clusters according to their species within the NJ trees (e.g. U33/U51 U14-1/U14-2 and U14-3/U14-4, snoR13/U18). While the variation within some species appeared to be low to moderate, variation in other species seemed unexpectedly high, with samples from the same species being placed in very different positions in a NJ tree. For example, S. flavus showed no within species variation apart from when the primer combinations U31/U51, U33/U51 and U14-3/U14-4 were used, which generated low variation in the species. Similarly, S. madagascariensis showed only moderate variation with the primer combinations U33/U51, U14-1/U14-2, U14-3/U14-4 and snoR13/U18. In contrast, all primer combinations generated high levels of variation within S. glaucus. Within the S. squalidus group of species, variation between species seemed as high as within species, and variation was generated by all primer combinations except U33/U51.

Fragments shared between at least one parent and their hybrid were generated by all primers except snoR2/snoR77Y (Table 4.1). For example, all fragments generated in

S. flavuswere also present in S. mohavensisfor all primer combinations except U33/U51

and U14-3/U14-3. For these last two primer setsS. flavuspossessed some fragments that were not found in S. mohavensis. As expected, tetraploid S. mohavensis contained additional fragments to those found in diploid S. flavus, however these additional bands could not be assigned clearly to its other diploid parent,S. glaucus, because this species was highly variable in fragment profile and only a few samples of it were examined. Within the S. squalidusgroup of species, which consisted predominantly of hybrids and their parents, fragments were shared between species. However, due to a lack of fragments that were exclusively shared between parents and hybrids, it was difficult to identify bands that could be used satisfactorily for hybrid detection. Within this group S.

squalidus was both a parent of a hybrid (i.e. S. cambrensis), and also a hybrid itself (i.e.

Chapter 4 Results

squalidus (10) shared more bands with its hybrids (S. cambrensis and S. teneriffae),

whereas the other sample ofS. squalidus(3) was closer in fragment type to its parents (S.

aethnensisandS. chrysanthemifolius). Most primer combinations generated fragments in

hybrids that were not present in their parents and vice versa. Thus, hybrid detection using snoRNA markers is likely to be more successful if based on shared fragments than on genetic distances computed from fragment profiles.

In the diploidSeneciospecies tested, it was estimated that the number of copies of each snoRNA cluster (Figure 4.1) were as follows (Table 4.1): at least three copies of cluster A (based on five bands generated by U31/U51 and U33/U51), two copies of cluster D (based on two U49/snoR2 bands of 410 and 550 bp), three copies of cluster E (five bands generated by snoR13/U18), two copies of cluster F (three bands generated), two copies of cluster G (three bands generated) and three copies of cluster J (five bands generated by snoR37/snoR22 in S. squalidus). Cluster B consisted of homologous genes only and the number of gene cluster copies could not be estimated. However, the primer combination U14-3/U14-4 generated six different fragments, the same number of fragments was obtained by reverse ePCR forA. thalianasuggesting a gene copy number similar to this species.

Chapter 4 Results

Table 4.1: Summary of radioactively labeled fragment analysis results. 14 primer combinations showed PCR amplification. Amplification success is indicated by the number of samples amplified/number of samples tested. + = present; - = absent. SR = snoR. between species within species/ species group* U31/U51 14/15 5 + +/+ + U31/U33 27/28 3 + -/+ + U33/U51 28/28 5 + +/- + U14-1/U14-2 24/28 2 + +/+ + U14-3/U14-4 26/28 6 + +/+ + U49/SR2 15/28 2 + +/- + SR2/SR77Y 4/28 2 - -/- - SR13/U18 25/28 5 + +/+ + U18/U54 24/28 2 + +/+ + F U61/SR14 14/15 3 + -/+ + G SR29/SR30 27/28 3 + +/+ + SR37/SR22 15/15 9 + +/+ + SR22/SR23 25/28 4 + +/+ + shared parents/hybrid fragments A B

Cluster Primer pair Amplification success Max. number of bands in diploids D E J Variation

*S. squalidusgroup includingS. vulgaris,S. vulgarisvar.hibernicusandS. teneriffae