Métodos Anticonceptivos

All specific U14 primers (Table 6.2) together with the U14-1 primer were tested for PCR amplification using one sample ofS. aethnensis, two ofS. chrysanthemifoliusand one of

S. squalidus. The expected band of about 80 bp was obtained from all reactions (not

shown). Furthermore, some primers, e.g. U14-2.2, U14-2.3 and U14-2.4, amplified additional bands of about 250, 450 and 750 bp in length, respectively. However, differences in banding patterns were evident between different variants (a, b and c; Table 6.2). For example, while primer U14-2.3a produced strong bands of 250 and 750 bp in

bothS. chrysanthemifoliussamples, primer U14-2.3b amplified a different band of about

450 bp in length. Unsurprisingly, all three of these bands were generated by the U14-2.3 primer. There were also differences in amplified band patterns between samples and species with both S. chrysanthemifolius samples exhibiting stronger band amplification

Chapter 6 Results than was the case in either of the other species. Additionally, bands present in both samples of S. chrysanthemifolius were sometimes absent from one or both of the other species. For example, only one band was generated by the U14-2.3 primers in S.

aethnensisandS. squalidus.

In summary, the U14-1/U14-2 universal primer pair produced sequences of multiple gene copies (at least nine in one diploid), but it was not possible to distinguish between alleles, paralogues and orthologues of the U14 gene. Furthermore, the sequences obtained from different samples/species were intermixed into four different groups resolved by PCO analaysis. These four groups did not distinguish species from each other, but rather identified four different types of sequence. Thus, sequences of U14 genes placed within one particular group were either identical or very similar to each other, whereas those placed in different groups differed considerably from one other. Some intergenic regions were found to be relatively long and to accommodate the snoR99 box H/ACA gene. Specific primers were designed and amplified successfully some samples, but were not further investigated because of the high complexity of this gene cluster. Further examination of this cluster would be time consuming and was, therefore, not possible within the scope of this thesis.

6.3.3 U61/SnoR14

6.3.3.1 Sequence generation from original universal primers

Sequences generated by the U61/SR14 primer pair were obtained from three species (S.

aethnensis,S. chrysanthemifoliusand S. squalidus) after pooling eight to ten samples per

species. The initial data matrix comprised 89 sequences, i.e., 38 from S. aethnensis, 12

fromS. chrysanthemifoliusand 39 fromS. squalidus, with each sequence being 137 bp in

length. Identical sequences were removed to leave 46 sequences in the alignment (Figure 6.9). The intergenic region (71 bp) contained 36 variable sites, while the two gene regions (66 bp) had seven variable sites. Only one indel (1 bp) was present in the snoR14 gene region, while several indels up to a maximum length of 12 bp were evident in the

Chapter 6 Results intergenic sequence. Interestingly, the indel of the snoR14 gene was present in its box C, which showed higher variability than other parts of the same gene sequence (Figure 6.9).

10 20 30 40 50 60 70 80 90 100 110 120 130

. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .

squac20 ACCCTCTAAGAAGTTCTGAGCGATTACCTTCTCTTATTTTTCTA-TTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

squac10 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCAAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

squac25 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

squac27 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCACTGA

squac31 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAG-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

squac9 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTA-TTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

chrysc11 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTTTTATTTTTTTA-TTTTTTTTTCAAATTTATTAAATT---TATAAAAAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCACTGA

chrysc1 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTTTTATTTTTTTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

chrysc2 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTTTTATTTTTTTA-TTTTTTTTTCAAATTTATTAAATT---TATAAAAAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

chrysc7 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTTTTATTTTTTTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

chrysc8 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc14 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCACTGA

aethc15 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAGA-GCGAGGTAA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc21 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc23 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTA-TTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc37 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTATTTTTTTTTTCAAATTTATTAAATT---TATAAAAAG-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc40 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTCTTATTTTACTAATTTTTTTTTCAAATTTATTAAATT---TATAAAAAA-GCGAGGTGA-GATAAAATTCAATGGTCTGTCAATCCCCTGA

squac14 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTTTCAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

squac28 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTTTCAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCACTGA

squac34 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTT-CTAATTTTTTTT-CAAATTTATTAAATT---TATAGAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

squac39 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTAAATT---TATAGAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

squac3 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

squac41 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTAAATT---TCTAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

squac42 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTT-CTAATTTTTTTT-CAAATTTATTAAATT---TATAGAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

chrysc3 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc12 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTT--CTAATTTTTTTTTCAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCACTGA

aethc20 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTTTCAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCACTGA

aethc29 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc2 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc34 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTT----TTTTTCTAATTTTTTTTTCAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc38 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTTTCAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc39 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTTTCAAATTTATTGAATT---TATAAAAA--GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc4 ACCCTCTAAGAAGTTCTGAGCGATTACTTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTAAATT---TATAGAAAA-GCGGGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc5 ACCCTCTAAGAAGTTCTGAGCGATTACCTTTT----TTTTTCTAATTTTTTTT-CAAATTTATTGAATT---TATAAAAAA-GCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCACTGA

squac12 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GTAAAAAGAAAGCGAGGTGACAATAAAATTCAATGGTCTGTCAATCCCCTGA

squac30 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GTAAAAAGAAAGCGAGGTGACAATATAATTCAATGGTCTGTCAATCCCCTGA

squac38 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GAAAAAAGAAAGCGAGGTGACAATAAAATTCAATGGTCTGTCAATCCCCTGA

chrysc10 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GTAAAAAGAAAGCGAGGTGACAATAAAATTCAATGGTCTGTCAATCCCCTGA

chrysc13 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GTAAAAAGAAAGCGAGGTGACAATAAAATTCAATGGTCTGTCAATCCACTGA

aethc11 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GTAAAAAGAAAGCGAGGTGACAATAAAATTCAATGGTCTGTCAATCCACTGA

aethc28 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GTAAAAAGAAAGCGAGGTGACAATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc7 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GTAAAAAGAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc8 ACCCTGTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTCTTTTT---GAAAAAAGAAAGCGAGGTGACAATAAAATTCAATGGTCTGTCAATCCCCTGA

aethc1 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTTCTTTTTGTAAAAAAAATAAAAAGAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCACTGA

aethc3 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTTGTAAATATTTGTTTTTTTTCTTTTTGTAAAAAAAATAAAAAGAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCACTGA

aethc6 ACCCTCTAAGAAGTTCTGAGCAATCATTTATTATATCTTTTCTTCGTAAATATTTGTTTTTTTTCTTTTTGTAAAAAAAATAAAAAGAAAGCGAGGTGACGATAAAATTCAATGGTCTGTCAATCCCCTGA

D’ C D C B A

Specific primer site

Figure 6.9: Alignment of 46 different U61/snoR14 sequences detected across three Seneciospecies. Conserved positions are shaded. The red part of the specific primer site symbol indicates the extension of the original primer. Coloured vertical bars indicate the clusters (A, B and C) to which the sequences belong to in the PCO plot and NJ tree (see below). Dotted line = antisense element; black line = gene region; red line = intergenic region. aeth =S. aethnensis, chry =S. chrysanthemifolius, squa =S. squalidus.

Chapter 6 Results

6.3.3.2 U61/snoR14 gene copies and organisation

Three well separated groups of U61/snoR14 sequences were detected by means of PCO analysis (Figure 6.10). Sequences obtained from each species (S aethnensis, S.

chrysanthemifoliusandS. squalidus) were present in each of these three different groups.

PCO 1 (66.26 %) P C O 2 (1 6 .3 8 )

S. aethnesis S. chrysanthemifolius S. squalidus

PCO 1 (66.26 %) P C O 2 (1 6 .3 8 )

S. aethnesis S. chrysanthemifolius S. squalidus

Figure 6.10: PCO plot of 46 different U61/snoR14 sequences detected in three species of Senecio. Within the blue encircled group, sequences from S. aethnensis are covered by those fromS. squalidus.

In the NJ tree (Figure 6.11), two well supported clades (100 %) were resolved, which corresponded to the blue and the green/red encircled groups in the PCO plot (Figure 11). The mean genetic distances between clades were: A vs B = 0.028, A vs C = 0.381, B vs C = 0.334. Molecular variance among the three clades (A, B and C; Figure 6.11) was much greater (82 %) than within clades (18 %). Interestingly, sequences of a certain length are present in certain clades (Figure 6.11). For instance, clade C contained only long sequences of 121 and 131 bp, whereas clades A and B contained shorter sequences of 117 to 120 bp, and 112 to 116 bp, respectively. The size differences in sequence between the clades A and B were mainly due to a four bp deletion (at positions 32 to 36 in the alignment, Figure 6.9) present in all sequences placed in clade B. Within each of the three clades sequences from the different species were very similar and intermixed (Figure 6.10 and Figure 6.11).

Chapter 6 Results 0.02

100

C (121 bp)

C (131 bp)

B (113-116 bp)

A (117-120 bp)

Figure 6.11: NJ tree constructed for 46 different U61F/snoR14R sequences obtained from three Senecio species (S. aethnensis, S. chrysanthemifolius and S. squalidus).

The same colours and symbols as in the PCO plot were used to identify sequences of different species. (S. aethnensis = ●, S. chrysanthemifolius = ■ and S. squalidus = ♦). Bootstrap support of the two main clades (A/B vs C) is indicated and lengths of

Chapter 6 Results By examining both sequence and fragment analysis data it was established that each sample had at least one fragment of sequence length found in clade C (121 and 131 bp, respectively), and fragments of sequence length found in clades A and/or B. Therefore, it is feasible that clades A and B contain sequences representing different allelic variants rather than different paralogous gene copies. Thus, clade specific primers might be required to provide further insights into the different copies of each gene.

6.3.3.3 Design of clade/gene specific primers

Seven different primers were designed by 3’ elongation of the original U61 universal primer (see alignment Figure 6.9). These primers consisted of one long and one short variant (e.g. U61Fc1l and U61Fc1s) for amplifying sequences assigned to each of the three clades in the NJ tree (Fig. 5.11), and an additional primer (U61Fc1_2) for amplifying sequences belonging to clades A and B. These specific primers ranged from 24 to 37 nt in length, had TMs from 48.8 to 62.2 °C, and GC contents from 28 to 44.4 % (Table 6.3).

Table 6.3: Sequences and characteristics of specific primers designed to amplify U61/snoR14 snoRNA gene cluster sequences. The first primer listed is the original primer sequence.

Name Direction Sequence (5' - 3') Length (nt) TM (° C) GC (%)

U61F TACACWACCCTCTAAGAAGTTCTG 24 54 41.7 U61Fc1l ACCCTCTAAGAAGTTCTGAGCGATTACCTTTTYTTA 36 61-62.2 36.1