A molecular cytogenetic analysis of chromosome behavior in Lilium ...

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Chapter 2on the other, two types of translocations can be distinguished: nonhomologous andhomoeologous translocations.During the past several years, a large number of polyploids have been induced by usinghybrids of species and cultivars of Lilium and the resulting neopolyploids were analysedthrough GISH (Barba-Gonzalez et al. 2004; Barba-Gonzalez et al. 2005b; Barba-Gonzalez etal. 2006b; Karlov et al. 1999; Khan et al. 2009a; Lim et al. 2000; Zhou et al. 2008b). For thesynthesis of polyploids, both somatic chromosome doubling of the F1 hybrids throughchemicals such as colchicine or oryzalin as well as sexual polyploidization throughnumerically unreduced (2n) gametes were used. These neopolyploid progeny are ideallysuitable for cytological analysis using GISH technique for two important reasons. 1. Thechromosomes of Lilium species are very large and suitable for cytological analysis. 2. Thegenomes of the parents used for producing hybrids and their neopolyploids are so welldifferentiated that structural rearrangements, if any, can be identified accurately throughGISH in meiotic as well as somatic cells. The main aim of the present study is to investigate,through GISH analysis, whether chromosomal rearrangements occur in the neopolyploids ofLilium. Furthermore, the reasons why intergenomic recombination in hybrids might bemistaken for chromosomal rearrangements are discussed.Materials and methodsPlant materialsPlant material consisted of polyploids derived from the hybrids of four groups of diploid (2n= 2x = 24) cultivars, viz., Longiflorum (L), Asiatic (A), Oriental (O) and Trumpet (T).Because the cultivars are derived from crossing some closely related Lilium species (McRae1998), the specific names of individual species are avoided and the letters in each caseindicate the genomes. The first three of these groups (L, A and O) have resulted from crossingof closely related species within each of the three taxonomic sections, viz., Leucolirion,Sinomartagon and Archelirion respectively. The last one, the Trumpet group, also belongs tothe section Leucolirion, the same as Longiflorum, but forms a separate crossability groupwithin the section and possesses a clearly differentiated genome (Lim et al. 2008a). For theanalysis of polyploids derived from somatic chromosome doubling, the progeny of a crossbetween an allotriploid ‘Triumphator’ (LLO) with an allotetraploid (LLTT) the latter suppliedby one of the Dutch lily companies (Worldbreeding BV) were used. The triploid parent of thiscross was produced by backcrossing the allotetraploid, LLOO, hybrid with diploidLongiflorum (LL). Meiotically doubled polyploids were produced by backcrossingLongiflorum × Asiatic (LA) and Oriental × Asiatic (OA) F1 hybrids with Asiatic parents inwhich the F1 hybrids had contributed 2n gametes and the resulting progenies were triploids(Barba-Gonzalez et al. 2006a; Khan et al. 2009a). Part of the backcross progeny of meiotic18
Chromosome rearrangements in Lilium hybridspolyploids was supplied by the following Dutch lily breeding companies: De Jong Lelies BV,Royal Van Zanten BV, Testcentrum BV, Vletter and Den Haan BV and Worldbreeding BV.Mitotic and meiotic chromosome preparationsFor mitotic chromosome preparation, young roots were treated with 0.7mM cyclohexamidefor 4-6 hours at 4°C then transferred to Carnoy’s Solution (Ethanol 3: Acetic acid 1) andstored at 4°C until use. Root tips were incubated in enzyme mixture (1% cellulose RS, 1%Pectolyase Y23, in 2mM citrate buffer, pH 4.5) for 90 minutes at 37°C. Mitotic metaphasechromosomes were spread according to Ross et al. (1996). For meiotic chromosomepreparation, young anthers with stages from prophase I to telophase II were collected andfixed in fresh Carnoy’s solution for 24 h at 4°C. Part of fixed anthers was squashed in a dropof 2 % acetocarmine to determine appropriate meiotic stage. Anthers with proper meioticstages were incubated in enzyme mixture containing 1% pectolyase Y23, 1% cellulase RS and1% cytohelicase in 10mM citrate buffer (pH 4.5) at 37 °C for about 25 – 35 minutes.Subsequently, the procedure used for meiotic chromosome preparations was the same as usedfor mitotic chromosomes.GISH procedureIn case of LLO × LLTT population, total genomic DNA was extracted from young leaves ofOriental cultivar ‘Sorbonne’ and Trumpet cultivar ‘Royal Gold’ with CTAB method. TheDNA was sonicated to 1-10kb fragments and used as probe. The DNA of Longiflorumcultivar ‘White Fox’ was autoclaved to 200-600bp fragments and used as block. For LA × AAand AA × OA hybrids and interspecific F1 genotypes, sonicated DNA from Longiflorumcultivar ‘White Fox’ and Oriental cultivar ‘Sorbonne’ was used as probe respectively, whileautoclaved DNA from Asiatic cultivar ‘Connecticut King’ was used as block. Probe DNAwas labelled with either Digoxigenin-11-dUTP or Biotin-16-dUTP by standard Nicktranslation according to the manufacturer’s instruction (Roche, Germany). The GISHprocedure was carried out as described previously (Khan et al. 2009a; Lim 2000). Briefly, thehybridization mixture contained 50% formamide, 10% dextransulphate, 2×SSC, 0.25% SDS,0.6-1.0 ng/μl for each probe and 15-50 ng/μl block DNA. After hybridization and stringencywashing, the probes labelled with Digoxigenin-11-UTP and Biotin-16-UTP were detected byanti-digoxigenin and Cy3-streptavidin systems respectively. Then the slides werecounterstained with 1 μg/ml DAPI and mounted with Vectashield. Preparation were analysedusing a Zeiss Axiophot epifluorescence microscope and photographed with a Canon digitalcamera.Chromosome identification and karyotypingImages of mitotic metaphase chromosomes were measured using the computer programMicroMeasure (Reeves and Tear 2000). In each of the four genomes (L, A, O, T), the19
Page 3 and 4: A molecular cytogenetic analysis of
Page 5: Table of ContentsChapter 1General I
Page 8 and 9: Chapter 1LilyLilies belong to genus
Page 10 and 11: Chapter 1counterparts (Finnegan 200
Page 12 and 13: Chapter 1renewed interest in detect
Page 14 and 15: Chapter 1recombination, mechanisms
Page 16 and 17: Chapter 1is only an equational segr
Page 18 and 19: Chapter 1quite divergent with vario
Page 21 and 22: Chapter 2An assessment of chromosom
Page 23: Chromosome rearrangements in Lilium
Page 27 and 28: Chromosome rearrangements in Lilium
Page 37 and 38: Chapter 3Elucidation of intergenomi
Page 39 and 40: Intergenomic recombination and chro
Page 49: Intergenomic recombination and chro
Page 52 and 53: Chapter 4AbstractMeiotic abnormalit
Page 54 and 55: Chapter 4There are some criteria to
Page 56 and 57: Chapter 4FISH experiments were perf
Page 58 and 59: Chapter 4was apparently shorter, wi
Page 60 and 61: Chapter 4bridges is explained as U-
Page 62 and 63: Chapter 4of the sexual polyploidize
Page 64 and 65: Chapter 4fragment have the same len
Page 66 and 67: Chapter 5AbstractSupernumerary (B)
Page 68 and 69: Chapter 5their origin, the structur
Page 70 and 71: Chapter 5Fig. 5.1. Discovery of B c
Page 72 and 73: Chapter 5In order to investigate th
Page 74 and 75:
Chapter 5Centric breakage and fusio
Page 76 and 77:
Chapter 5It has not, however, been
Page 78 and 79:
Chapter 6The results presented in t
Page 80 and 81:
Chapter 6model for molecular cytoge
Page 82 and 83:
Chapter 6Fig. 6.2. The meiosis proc
Page 84 and 85:
Chapter 6over events during FDR mei
Page 86 and 87:
Chapter 6been detected with GISH an
Page 88 and 89:
Chapter 6Another feature caused by
Page 91 and 92:
ReferencesAbe, H.A., Nakano, M.N.,
Page 93 and 94:
ReferencesChen, Q., and Armstrong,
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ReferencesHartlerode, A.J., and Scu
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ReferencesLarson, S.R., Kishii, M.,
Page 99 and 100:
ReferencesMcClintock, B. 1931. Cyto
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ReferencesRai, R., Zheng, H., He, H
Page 103 and 104:
ReferencesStewart, R.N. 1947. The m
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ReferencesZhang, L., Pickering, R.,
Page 108 and 109:
Summarychromosome rearrangements. T
Page 111 and 112:
SamenvattingLelie (Lilium) is in de
Page 113 and 114:
Samenvattingaantal 35 met daarnaast
Page 115 and 116:
摘要百合系百合科百
Page 117 and 118:
Acknowledgements淡看世事去
Page 119:
Curriculum VitaeSonglin Xie was bor
Page 123:
Education Statement of the Graduate
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