Actividades

Differences within the five plastomes mostly depend on repeat structures, at least in non- coding regions. PGI may therefore also reflect a result of structural evolution of the plastid chromosome. Furthermore, since the five plastomes are perfectly syntenic, a unique opportunity for a comparative analysis of indels and sequence repetition is given. Such an analysis is usually difficult between distant species.

3.2.2.1.1.Indels within the five plastomes

Insertions, deletions and repetitions are relatively frequent within the five plastomes. Relative to plastome IVatro, 1456 nucleotide insertions and 3819 deletions are found in plastome Ijoh, for plastome IIsuavG, IIIlam and Vdou1 numbers are 1156/2598, 1701/3561, 864/2557, respectively. As expected, indels occur less often in genes and are present in only relatively few polypeptide genes. Only accD, clpP, ndhD, ndhF, rps18, rpl22, ycf1 and ycf2 are affected. These changes in genes will be discussed separately (Chapters 3.2.3.2.1 - 3.2.3.2.4).

3.2.2.1.2.Tandem and palindrome repeats

Both repeat types, tandem and palindrome repeats, are distributed highly similar between all five plastomes. In terms of tandem repeats, on average 61 were detected, within a range of 55 (plastome IVatro) up to 70 (plastome Ijoh). The mean copy number was 4.5 copies per tandem and an average size of 41 bp per copy. In all plastomes the largest tandem repeat regions spanning more than 1 kbp were found in the two ycf2 genes in the IR. They consist of variants of an AAG/TTC trinucleotide sequence. Expanded tandem repeats are also frequent and overlapping in accD and ycf1 contributing to the substantial sequence divergence of these genes (Chapters 3.2.3.2.3 and 3.2.3.2.4 ). Associated with small identical tandem repeats in all five plastomes are rpl32, ndhF and the tandemly repeated trnfMCAUI and trnfMCAUII. More

interesting are tandem repeats, which change coding potential specific to distinct plastomes. These repeats, generally of moderate size and low copy number, are found in clpP, ndhF and

rps18 (Chapters 3.2.3.2.1 and 3.2.3.2.2). The plastome-specific repeat differences associated with ccsA, rpl22, rpl32, rps19 and trnSGCU do not change coding context, since the differing

Figure 20. Overall distribution of sequence divergence and repetition in the Oenothera plastid chromosomes. Repeat regions and areas of high divergence are often, but not always, correlated.

Analyses of palindrome repeats give similar results. Approximately 70 palindrome repeats on average, with a maximal gap size of 3 kb or less, were detected within the five plastome sequences. However, these repeats are generally smaller and far less variable. Sizes ranged from 32 bp (detection limit given by the threshold applied) up to 56 bp. They were detected in

accD, ccsA, matK, ndhD, ndhF, ndhJ, petD, psaA, psaB, psbH, rpl32, rpoA, rpoB, rps18,

ycf1, ycf2 and ycf4, but no notable change of coding sequence associated with palindrome repeats was observed. The highly polymorphic genes accD and ycf2 are exceptions, as it is

ndhD. Here, frame changing insertion of a single nucleotide in a polyA-tail (Chapter 3.2.3.2.2) is located in a palindrome repeat. Presence and absence of repetitive elements and divergent regions correlate well, but repeat content and high divergence are not strongly linked (Figure 20).

3.2.2.2.The large 56 kbp inversion

regions between trnQUUG/accD and rps16/rbcL are highly divergent and contain palindromes

as well as tandem repeats. Therefore, a closer investigation of the breaking points in the

Oenothera plastomes was performed. This analysis also included the study of the ancestral situation in the subsection Munzia, lacking the inversion (Gordon et al., 1982; Hachtel et al., 1991; Systma et al., 1993; Hupfer et al., 2000).

Figure 21. Scheme indicating the 56 kbp inversion that occurred in the sequence intervals rps16/rbcL and trnQUUG/accD, respectively, in the large single copy segment of the Oenothera plastid

chromosomes. The inversion flanked by trnQUUG and rbcL changes the orientation of 35 genes.

Transcription of genes above lines is counter clockwise, below lines clockwise.

In summary, within the Oenothera plastomes more tandem repeats are detected in the

trnQUUG/accD spacer, than in the rps16/rbcL spacer. In detail, 5 to 8 tandem repeats were

detected between the trnQUUG and accD spacer depending on the plastome. Only a maximum

of two tandem repeats were found in plastome IVatro and none in plastome Vdou1 in the

detected in all five plastomes. Their number varies enormously, between one (plastome Vdou1) and 17 (plastome IIIlam). Furthermore, the same sequence motifs can be present in tandem and palindrome arrangement. The repetition patterns preclude an accurate demarcation of the insertion breakpoints and also affect the highly variable N-terminal region of AccD (Chapter 3.2.3.2.4).

In an attempt to better understand the inversion breakpoints and underlying processes, the corresponding regions in Oe. villaricae strain berteriana Schwemmle, a member of the closely related sister subsection Munzia, were sequenced (rps16/trnQUUG, accession no. EU255777

and rbcL/accD, accession no.EU255778). Subsection Munzia lacks this inversion (Hachtel et al., 1991). The berteriana Schwemmle regions corresponding to the Oenothera breakpoints between rps16/trnQUUG and rbcL/accD do not display the pronounced divergence. The spacer

region between rps16 and trnQ in that plastid chromosome lacks tandem and palindrome repeats. Nevertheless, the entire region is conserved and present in two parts in all five

Oenothera plastomes, separated by an interspersed Oenothera-specific sequence interval. The berteriana Schwemmle region between rbcL and accD, in turn, lacks palindromes, but contains two tandem repeats. Approximately 1.5 kb of the berteriana Schwemmle rbcL/accD spacer are unique to the berteriana Schwemmle plastome. Conversely, trnQ/accD spacer sequences between 1.5 and 2.5 kb depending on the Oenothera plastome have no equivalent in the berteriana Schwemmle plastome. The same holds true for the Oenothera rps16/rbcL spacer, in which the number of unique nucleotides differs between approximately 50 and 500 bp. The repeat structure in the rps16/rbcL spacer of the Oenothera plastomes (Figure 20) appears to be linked to the inversed arrangement of rbcL and the result of duplication and relocation during the inversion process, as the berteriana Schwemmle equivalent is missing. As mentioned, palindrome repeat copies split among both spacers are not rare in the

Oenothera plastomes, but no such cases were detected in berteriana Schwemmle with the selected threshold.