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126 6. DO ANY SPECIES BOUNDARIES.... of polymorphic loci were the most effective, followed by RAPDs, and enzymes (Figure 6.1). High efficiency was also noted for SSR markers although these results could be somehow biased because only primers that had revealed high polymorphism in the studies of Jones et al. (2001) were chosen for the present analyses. Out of seven pairs of SSR primers tested, five (71%) detected polymorphic loci. Two primers produced single-locus amplification patterns, and three others detected duplicated loci (Table 6.6). In total six polymorphic loci were observed. Dominant mode of inheritance with null alleles was characteristic of SSR loci with (CA) n repeated motifs, whereas codominance was observed for loci with the other motifs. Single band alleles were observed for A02, A11, and C11 (Figure 6.2). At two SSR loci A10 and H06 alleles with a different number of bands were observed. At the former an allele, A10-1a had two bands whereas another had three bands (A10-1b). At the H06 SSR locus two- and single band alleles were found. Heterozygotes at these two loci were expected to have five (A10) or three (H06) bands however, the middle bands were very often faint or even not visible. The most dramatic findings involved total uselessness of 13 STS markers either derived from L. perenne or from cereals, H. vulgare, and A. sativa. Although all primer pairs were selected on the basis of earlier studies (Taylor et al. 2001), in which they amplified a single product, in the present work optimised PCR conditions resulted in the amplification of a single product of the expected size only for 50% of sequences. Expectedly, the majority of such sequences were derived from L. perenne (LOLPISO5A, LOLPISO5B, ASNL, Trx) but also from H. vulgare (AS1) and A. sativa (CDO1508). The other seven primer sets could not be optimised to amplify a single band however, two strong reproducible bands were obtained in a case of four primer sets derived from L. perenne (LOLLOPIB, Gln2), H. vulgare (BCD450) and A. sativa (CDO504) genes. Difficulties were encountered using primers developed from
6. DO ANY SPECIES BOUNDARIES... 127 L. temulentum gene, LTLHAB, and two H. vulgare genes, ASN and HS1. They all amplified several bands. Nevertheless, these multiple products were highly reproducible in parents and F 2 individuals used for screening. Unfortunately, all STS markers were monomorphic in both parents and F 2 progeny and thus they were not useful in mapping studies. This was even though a wealth of methods was applied to disclose polymorphism. Amplification polymorphism was analysed for those primer sets that gave multiple but stable products. PCR products for single-band STS markers were analysed for restriction site polymorphism. In a case of two-band STS markers, bands were eluted from gels, re-amplified and then PCR products were subjected to restriction analyses. In comparison with STS markers derived from Poaceae, primer sets based on M. tuberculosis genes proved to be quite effective in disclosing polymorphism between parents
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This research was done within the E
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Acknowledgements I would like to th
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8 CONTENTS 4.4.4. Role of transposo
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10 CONTENTS 9.3.2. Similarity of ge
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12 1. INTRODUCTION
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14 1. INTRODUCTION occasionally use
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16 1. INTRODUCTION On the basis of
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18 1. INTRODUCTION The evidences fr
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20 1. INTRODUCTION overlap between
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22 1. INTRODUCTION et al. 2000; Cat
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24 1. INTRODUCTION According to the
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26 1. INTRODUCTION 1.6. APPLICATION
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28 1. INTRODUCTION of evolution wit
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30 1. INTRODUCTION are continuously
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2. GOALS The aim of this research w
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3. MORPHOLOGICAL VARIATION OF L. MU
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36 3. MORPHOLOGICAL VARIATION... fo
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38 3. MORPHOLOGICAL VARIATION...
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40 3. MORPHOLOGICAL VARIATION... Wi
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42 3. MORPHOLOGICAL VARIATION... va
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44 3. MORPHOLOGICAL VARIATION... 3.
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46 3. MORPHOLOGICAL VARIATION...
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48 3. MORPHOLOGICAL VARIATION... in
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50 3. MORPHOLOGICAL VARIATION... pl
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52 3. MORPHOLOGICAL VARIATION... di
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4. GENETIC DIVERSITY OF L. MULTIFLO
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56 4. GENETIC DIVERSITY... deed con
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58 4. GENETIC DIVERSITY... 4.2.2. D
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60 4. GENETIC DIVERSITY...
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64 4. GENETIC DIVERSITY... RAPD and
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66 4. GENETIC DIVERSITY... SSAP pol
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68 4. GENETIC DIVERSITY... It can b
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72 4. GENETIC DIVERSITY... than wit
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176 7. ROLE OF QTLs IN THE EARLY EV
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196 8. MOLECULAR PHYLOGENY OF THE G
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9. PHYLOGENETIC RELATIONSHIPS BETWE
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224 9. PHYLOGENETIC RELATIONSHIPS..
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10. MARKER EFFICIENCY 10.1. INTRODU
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238 10. MARKER EFFICIENCY 10.3. DNA
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240 10. MARKER EFFICIENCY method. H
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242 10. MARKER EFFICIENCY alleles p
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244 10. MARKER EFFICIENCY proportio
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246 10. MARKER EFFICIENCY 10.7. CON
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248 11. CONCLUSIONS ABOUT EVOLUTION
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250 12, LITERATURE CITED Bączkiewi
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252 12, LITERATURE CITED Clayton WD
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254 12, LITERATURE CITED Giddings G
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256 12, LITERATURE CITED Jing R, Kn
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258 12, LITERATURE CITED MacDonald
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260 12, LITERATURE CITED Payne RC,
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262 12, LITERATURE CITED Schiex T,
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264 12, LITERATURE CITED United Sta
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266 12, LITERATURE CITED Zielinski
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268 13. SUPPLEMENTARY MATERIALS pla
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270 13. SUPPLEMENTARY MATERIALS
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274 13. SUPPLEMENTARY MATERIALS ANN
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280 13. SUPPLEMENTARY MATERIALS Ann
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290 13. SUPPLEMENTARY MATERIALS •
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292 13. SUPPLEMENTARY MATERIALS The
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296 13. SUPPLEMENTARY MATERIALS fie
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The average gene diversity between
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306 13. SUPPLEMENTARY MATERIALS Unb
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308 14. ABBREVIATIONS Lolcopia1 - L
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310 14. ABBREVIATIONS Drosophila wi
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312 14. ABBREVIATIONS Saccharomyces
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314 15. SUMMARY At different stages
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316 15. SUMMARY L. multiflorum mito
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REVIEWERS’ COMMENTS With a great
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