Sequence generation from original universal primers

From two samples of each of S. aethnensis, S. chrysanthemifolius, S. squalidus and S.

cambrensis, and one sample of S. vulgaris, 112 clones yielded 102 good quality

sequences of about 150 bp in length. These comprised 11 sequences ofS. aethnensis, 32

of S. chrysanthemifolius, 29 of S. squalidus, 8 of S. vulgaris, and 22 of S. cambrensis.

The number of different sequences identified in a particular sample ranged from two (in a sample of S. aethnensis) to eight (in a S. cambrensis sample). However, the maximum number of different sequences obtained for a diploid species (in samples of S.

chrysanthemifolius) was six, indicating that at least three copies of this gene combination

are present in diploidSenecio.

After removing all identical sequences, the alignment consisted of 21 different sequences having a maximum length of 153 bp. Of this, 57 bp comprised the intergenic region, and 96 bp comprised the gene region which included both primer sequences (Figure 6.2). The intergenic region, which is underlined in red in Figure 6.2, contained 19 variable sites and 5 indels, whereas the U51 gene region (3’ end gene; underlined in black in Figure 6.2) contained 8 variable sites and 1 indel. The U33 gene region (5’ end gene, underlined in black in Figure 6.2) contained only its primer sequence and therefore no variable sites were observed (Figure 6.2). It should be noted that both S. squalidus

samples and one individual ofS. cambrensisexamined contained sequences that differed in their box C sequences by one nucleotide from all other sequences.

Chapter 6 Results

10 20 30 40 50 60 7 0 80

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13c13 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTAA--AAAAAA-TAGTATTTGCAA 13c14 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTAA--AAAAAAATAGTATTTGCAA 13c1 CATGCACTACCATCTGATCTGTTTCTTTTGCTAATCAATCATCTCTTTTTTTTTTATTT--AAAAAAATAGTATCTGCAA 13c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTAA--AAAAAA-TAGTATTTGCAA 73c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCGTCTCTTTATTTGTATTTT---AACAAATAGTTTTTGCAA 74c5 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTATTTAAAAAAATAGTATCTGCAA 74c7 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTATTTAAAAAAATAGTATCTGCAA 74c8 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCGTCTCTTTATTTGTATTTT---AACAAATAGTTTTTGCAA 1c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTATTTGTATTTT---AACAAATAGTATTTGCAA 1c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTT----AAAAAATAGTATTTGCAA 46c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAAACAATCATCTCTTTTATCTATTTTA---AAAAAGTAGTTTTTGCAA 46c3 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTAA--AAAAAAATAGTATTTGCAA 46c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTTT---AACAAATAGTATTTGCAA 46c9 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGCATTTT---AACAAATAGCTTTTGCAA 4c1 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTA----AAAAAATAGTATTTGCAA 4c3 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTTT---AACAAATAGTTTTTGCAA 4c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTTT---AACAAATAGTTTTTGCAA 53c1 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTAA--AAAAAA-TAGTATTTGCAA 53c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTAT---AACAAATAGTTTTTGCAA 80c11 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTTTTGTATTTT---AACAAATAGTATTTGCAA 80c19 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTTT--CAAAAAATAGTATTTGCAA

90 100 11 0 120 130 1 40 150

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13c13 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 13c14 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 13c1 TTGATGATGCAATAATTTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 13c2 TTGATGATGCAATAATTTATTAATAATGAGATCATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 73c2 TTGATTATGCAATAATTTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 74c5 TTGATGATGCAATAATCTATTAATAATGAGATTATCCTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 74c7 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 74c8 TTGATTATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 1c2 TTGATGATGCAATAATTAATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 1c4 TTGATGATGCAATAATTAATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c2 TTGATGATGCAATAATTTATTAATAATGAGATTATCCTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c3 TTGATGATGCAATAATTTATTAATAATGAGAGCATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c4 TTGATGATGCAATAATCTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c9 TTGATGATGCAAAAATTAATAAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 4c1 TTGATGATGCAATAATTTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 4c3 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 4c4 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAAT-GATCACCATCTTTCGGCTGA 53c1 TTGATGATGCAATAATTTATTAATAATGAGAGCATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 53c4 TTGATGATGCAATAATTAATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 80c11 TTGATGATGCAATAATCTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 80c19 TTGATGATGCAATAATTAATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA

D

D

C

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13c13 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTAA--AAAAAA-TAGTATTTGCAA 13c14 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTAA--AAAAAAATAGTATTTGCAA 13c1 CATGCACTACCATCTGATCTGTTTCTTTTGCTAATCAATCATCTCTTTTTTTTTTATTT--AAAAAAATAGTATCTGCAA 13c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTAA--AAAAAA-TAGTATTTGCAA 73c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCGTCTCTTTATTTGTATTTT---AACAAATAGTTTTTGCAA 74c5 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTATTTAAAAAAATAGTATCTGCAA 74c7 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTATTTAAAAAAATAGTATCTGCAA 74c8 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCGTCTCTTTATTTGTATTTT---AACAAATAGTTTTTGCAA 1c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTATTTGTATTTT---AACAAATAGTATTTGCAA 1c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTT----AAAAAATAGTATTTGCAA 46c2 CATGCACTACCATCTGATCTTTTTCTTTTGCTAAACAATCATCTCTTTTATCTATTTTA---AAAAAGTAGTTTTTGCAA 46c3 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTAA--AAAAAAATAGTATTTGCAA 46c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTTT---AACAAATAGTATTTGCAA 46c9 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGCATTTT---AACAAATAGCTTTTGCAA 4c1 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTA----AAAAAATAGTATTTGCAA 4c3 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTTT---AACAAATAGTTTTTGCAA 4c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTTT---AACAAATAGTTTTTGCAA 53c1 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTT-TTTATTTAA--AAAAAA-TAGTATTTGCAA 53c4 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATTAATCATCTCTTTTTTTGTATTAT---AACAAATAGTTTTTGCAA 80c11 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTTTTGTATTTT---AACAAATAGTATTTGCAA 80c19 CATGCACTACCATCTGATCTTTTTCTTTTGCTAATCAATCATCTCTTTTATTTATTTTT--CAAAAAATAGTATTTGCAA

90 100 11 0 120 130 1 40 150

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13c13 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 13c14 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 13c1 TTGATGATGCAATAATTTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 13c2 TTGATGATGCAATAATTTATTAATAATGAGATCATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 73c2 TTGATTATGCAATAATTTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 74c5 TTGATGATGCAATAATCTATTAATAATGAGATTATCCTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 74c7 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 74c8 TTGATTATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 1c2 TTGATGATGCAATAATTAATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 1c4 TTGATGATGCAATAATTAATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c2 TTGATGATGCAATAATTTATTAATAATGAGATTATCCTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c3 TTGATGATGCAATAATTTATTAATAATGAGAGCATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c4 TTGATGATGCAATAATCTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 46c9 TTGATGATGCAAAAATTAATAAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 4c1 TTGATGATGCAATAATTTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 4c3 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 4c4 TTGATGATGCAATAATTTATTAATAATGAGATTATCTTTGATTAACTTAAAT-GATCACCATCTTTCGGCTGA 53c1 TTGATGATGCAATAATTTATTAATAATGAGAGCATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 53c4 TTGATGATGCAATAATTAATTAATAATGAGATTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 80c11 TTGATGATGCAATAATCTATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA 80c19 TTGATGATGCAATAATTAATTAATAATGAGAGTATCTTTGATTAACTTAAATTGATCACCATCTTTCGGCTGA

D

D

C

Figure 6.2: Alignment of 21 different U33/U51 sequences in Senecio. Conserved positions are shaded. Black line = gene region; black dotted line = antisense element; red line = intergenic region; C and D boxes are indicated (C and D). Sequence names refer to the sample and clone they were taken from. 1, 4 = S. aethnensis; 13 = S.

chrysanthemifolius; 73, 74 =S. squalidus; 46 =S. vulgaris; 53, 80 =S. cambrensis. Some

Chapter 6 Results

6.3.1.2 Gene copies and organisation

The different U33/U51 sequences identified fell into two clades (indicated by green and red bars) in the NJ tree (Figure 6.3). Both clades were structured containing several highly supported subclades (Figure 6.3). It was noted that almost all sequences of S.

vulgaris differed considerably, occupying single clades or sharing with S. cambrensis

only.Senecio cambrensis is the allohexaploid hybrid ofS. vulgarisand S. squalidus,and possessed all of the U33/U51 sequences found inS. vulgarisand some of those present in

S. squalidus. It was also the case that the homoploid hybrid species S. squalidus

contained all of the sequences found in one of its parents, S. chrysanthemifolius, and some of those present in its other parent,S. aethnensis(Figure 6.3).

All species except S. vulgaris were represented by two samples in this study. Rather surprisingly, sequences found within individuals were placed in different clades indicating that these sequences might represent different gene copies. However, more than two sequences obtained from samples of diploid species (e.g.S. chrysanthemifolius

(sample 13) and S. squalidus (sample 74)) were placed within the “green” clade suggesting more than one gene copy present in this clade. Interestingly, some sequences obtained from samples of the same species differ greatly whereas others show complete identity. For example, while the sequences of the two S. aethnensis samples differed considerably,S. cambrensissamples share four sequences (Figure 6.3).

Chapter 6 Results

Figure 6.3: NJ tree derived from different sequences generated by the U33F/U51R primer pair across five species of Senecio. Relationships are based on sequence variation using the Maximum Composite Likelihood method and pairwise deletion option for gaps/missing data. Bootstrap values are shown above or below branches. Sample identification numbers are given in brackets after species’ names. The coloured vertical bars indicate the different clades and therefore the putative gene copies present across the

Chapter 6 Results

6.3.2 U14-1/U14-2