Final Degree Project Biochemistry “Study of the evolution and the structure of the ribosomal RNA genes in plants: Analysis of the intergenic spacer (IGS) in a hybrid complex of species of the genus Armeria” Sònia Valle Durán Internal supervisor: Sònia Garcia Giménez External supervisor: Teresa Garnatje Roca June-July 2016 0 Cover picture: Armeria maritina: http://www.flickriver.com/photos/ken-ichi/4620446579/ 1 Abstract Hybridization and introgression phenomena are common in the genus Armeria. Armeria pungens is an example and it has suffered introgression with other pure species, Armeria macrophylla, which is very close to it. They have been found in the south of the Iberian Peninsula. In this TFG we have studied this phenomenon based on the IGS region of its genome, a non-coding region and thus highly variable and subject to evolution. So we have sequenced both two “pure” species and the hybrid and we have studied several elements located in this region, as well as its size and its correlation with the genome size. On the other hand we have analyzed another non-coding region of the genome, the ITS region, which is also highly variable and presents polymorphism in some nucleic bases depending on the taxon. All information indicates that introgression has worked in favour of Armeria macrophylla as the similarity of the hybrid is higher in many aspects in which we have deepened throughout the project. This research work is a part of a series of researches on the genus Armeria started in a previous TFG. The investigation has been extended with the analysis of more individuals and populations of the three taxa and the genome size. 2 3 INDEX Page 1. Introduction 1-2 1.1. Genus Armeria 1 1.1.1. Armeria pungens 1-2 1.1.2. Armeria macrophylla 2 1.2. Hybridization 3 1.3. Introgression 3 1.4. Chloroplast capture 3 1.5. Introgression phenomenon in the genus Armeria 4 1.6. Ribosomal DNA 5-6 1.7. Transposable elements (TE) 6 2. Hypothesis and objectives 7 3. Materials and methods 8-15 3.1. Materials 8 3.2. Methods 9-15 3.2.1. DNA extraction with DNeasyTM Plant Minikit 9 3.2.2. DNA extraction with the CTAB method 9-10 3.2.3. PCR: Polymerase chain reaction. DNA amplification 10 3.2.4. Sequencing of the entire IGS unit 11 3.2.5. Extraction and purification of DNA 11-12 3.2.6. Cloning 12-13 3.2.7. Flow cytometry 13 3.2.8. Sequence analysis and primer design 14-15 3.2.9. Statistical methods 15 4. Results 16-26 5. Discussion 27-30 6. Conclusion 30 7. Literature 31-35 4 0 1. Introduction 1.1. Genus Armeria Armeria Willd. is a genus of shrubby low-growing evergreen perennials (Figure 1). Although more than two hundred species have been described, only less than a hundred names are currently accepted. The genus was described by Carl Ludwig Willdenow, a German botanist from the XVIIIth century. It belongs to the Plumbaginaceae, a flowering plant family with a worldwide distribution (from arctic to tropical conditions), particularly associated with salt-rich steppes, marshes, and sea coasts. Most species are perennial herbaceous plants. The family includes 24 genera and about 800 species. Armeria species are usually found on coastlines and are mostly native to the Mediterranean. Armeria species (such as A. pungens and A. macrophylla) are strongly affected by natural hybridization and introgression (see page 4) (Nieto Feliner et al. 2001; Tauleigne-Gomes & Lefèbvre 2005; Piñeiro et al. 2011). 1.1.1. Armeria pungens Armeria pungens (Link) Hoffmanns. & Link is an herbaceous plant of genus Armeria that forms small shrubs reaching heights of about 80 cm. The stems are lignified at the base, robust and highly branched. Leaves are glabrous, linear to lanceolate, pointed. Flower heads (or capitula) are pale pink, gathered in globular inflorescences at the top of long pedicels (Figure 1). Figure 1. Armeria pungens http://www.bioscripts.net/herbarios/h_HFUS/pl g_178.html This small shrub grows in coastal sand-dunes and beaches, at about 0–1 meter above sea level and they are found in the southwest of the Iberian Peninsula (Figure 2). 1 Figure 2. Distribution of Armeria pungens. Some populations are also found in the northern coast of Corsica http://www.floravascular.com/index.php ?spp=Armeria%20pungens 1.1.2. Armeria macrophylla Armeria macrophylla Boiss. & Reuter is an herbaceous plant of genus Armeria. It is a perennial herb up to 70 cm. It has two types of leaves of about 10-27 cm: the light green and very narrow leaves with cespitose appearance and the gray -green narrow leaves up to 2 mm wide and covered with short and thin hairs. The pink flowers are grouped into capitula that grow at Figure 3. Armeria macrophylla the end of very long stems, up to http://www.florasilvestre.es/mediterranea/Plumb 60 cm. Each flower is wrapped in aginaceae/Armeria_macrophylla.htm white tissue that remains after the flower dries. The capitula are wrapped in straw-colored scales, hairless and membranous on the edge, and grow from the outside inward (Figure 3). It grows in coastal areas on sandy soils, specifically in the South Atlantic coast of the Iberian Peninsula (Figure 4). Figure 4. Distribution of Armeria macrophylla http://www.floravascular.com/index.php ?spp=Armeria%20macrophylla 2 1.2. Hybridization Hybridization is the process of interbreeding between two genetically different species (interspecific) or genetically divergent individuals from the same species (intraspecific). It results in a new cross breed or specie with a new genotype. This process is very common between plants leading to usually fertile progeny that may have advantageous characteristics with respect to the parental species. 1.3. Introgression It is a type of hybridization resulting from an interbreeding process known as backcrossing (Anderson & Hubricht 1938, Anderson 1949), the cross between an interspecific hybrid and one of its parental species. Introgression is more complex than simple hybridization because it results in a non-uniform mixture of the two parental genomes. This process increases the genetic variation of one or both of the parental species (Anderson 1949; Anamthawat-Jónsson 2001) and can contribute to their adaptation and speciation (Grant et al. 2005). 1.4. Chloroplast capture This is an evolutionary phenomenon tied to introgression that, after hybridization and subsequent backcrossings, results in a hybrid plant with a new combination: a chloroplast genome whose origin is from one parent and a nuclear genome whose origin is from the other parent (Grant et al. 2005). Chloroplast capture may explain the inconsistencies found in gene trees based on nuclear and chloroplastic markers in plants. Armeria pungens exhibits evidence for an ancient or ongoing introgression from sympatric congeners such as the sharing of ITS polymorphisms or the close relationship between plastid haplotypes (Piñeiro et al. 2011), suggesting chloroplast capture. 3 1.5. Introgression phenomenon in the genus Armeria As mentioned before, genus Armeria is subject to introgression as it is the case of Armeria pungens which has experienced this phenomenon with Armeria macrophylla resulting in a hybrid termed here as introgressed Armeria pungens (Figure 5). Armeria macrophylla Armeria pungens Introgressed Armeria pungens Figure 5. Introgression phenomenon in genus Armeria. The leaves of the introgressed taxa are only slightly shorter than the leaves of Armeria macrophylla but not as short as the leaves of Armeria pungens so the morphological distinction is difficult 4 1.6. Ribosomal DNA Ribosomal DNAs (rDNA) are the genes which code for ribosomal RNA that, along with proteins, form the ribosomes. Eukaryotic rDNA consists of a tandem repeat of a “large” operon composed of 18S, 5.8S and 28S (26S) rRNA genes and intergenic spacers (Figure 6). These regions are known as the nucleolus organizer regions, forming the nucleolus, expanded chromosomal loops where rDNA is transcribed. Figure 6. Eukaryotic rDNA https://en.wikipedia.org/wiki/Riboso mal_DNA#/media/File:Eucaryot_rd na.png There is another gene that codes for 5S rRNA also organized in tandem repeats in the genome in most eukaryotes, although it can be linked to the large rDNA operon as a single repetitive unit in certain groups (Garcia et al. 2009). Genes encoding ribosomal RNA and spacers are highly conserved among species and occur in tandem repeats that are thousands of copies long. Each one is separated by regions of non-transcribed DNA, termed intergenic spacer (IGS) or non-transcribed spacer (NTS) which are more variable due to insertions, deletions, and point mutations high enough to generate intra- and interspecific variability (Hillis & Dixon 1991; Baldwin et al. 1995) even between closely related species because of the low evolutionary pressures. The intergenic spacer (IGS) is the region of non-coding DNA between the rRNA genes 18S and 26S and can change very rapidly in evolution. Variability in the IGS can cause heterogeneity in rDNA repeat length among members of the same genus or species (Saghai-Maroof 1984; Rogers 1986). This variability is found in the number of subrepetitive elements within the IGS which are generally between 100 and 200 base pairs in length (Yakura 1984; McMullen 1986). These subrepetitive elements could have arisen from transposition events since they show some similarities with transposons, and it also appears that they are "hot spots" for recombination (Rogers 1986). It is also possible that subrepeats in plant intergenic spacers act as RNA processing sites or as transcription termination sites (De Winter 1986; Labhart 1986; Harrington 1987). 5 Chloroplasts also have their own rRNA genes, composed of 23S, 16S and 5S rDNA. rRNA gene sequences and some of their intergenic spacers (both nuclear and chloroplastic) are used for phylogenetic studies.