毛白楊未減數(shù)花粉發(fā)生及相關(guān)分子標記研究

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7、M?yr _T D F 2 4 #N*g 螿 pe 眰墊裇 u/f&T 鴙 sQg 卂蹚Nek xvz 0sQ.曂?踜vhg*g 螿 pe 眰墊苸迉 f:g6R RPh 皨NPSO 鵚瞼Occurrence and Molecular Markers of Unreduced Pollen in Chinese White Poplar(Zhang ZhenghaiDirected by Prof.Kang Xiangyang)AbstractThe production of unreduced pollen in Chinese white poplar (Populus tome

8、ntosaCarr.) has been reported. Studying the mechanisms of unreduced pollen was importantfor selection and utilization of unreduced pollen in triploid breeding. The objectiveof this research was to determine the mechanism of unreduced pollen formation inindigenous populations of Chinese white poplar.

9、 Occurrence frequency, cytologicalmechanisms and molecularmarkers involved in unreducedpollen production wereinvestigated by morphology of pollen grains, cytology of meiosis, AFLP and cDNA-AFLPmarkers.1. Pollen grains of 224 clones were investigated for the production of unreduced pollenbased on pol

10、len size difference. It was observed that six clones which from Henan andShannxi were subsequently determined to only produce normal pollen and the remainderproduce unreduced pollen at different frequency range from 0.6% to 21.9%. The analysisof variance on the frequency of unreduced pollen in Chine

11、se white poplar revealedsignificantdifferencesbetweenindigenouspopulationsandhighlysignificantdifferences among clones within indigenous populations.2. Microscopic examination was used to determine the second meiosis of pollen mothercells in Chinese white poplar. Two types of spindles (parallel spin

12、dles and tripolarspindles) were observed as indicated by the orientation of the chromosomes. So did dyadsand triads at the tetrad stage. Furthermore, indirect immunofluorescence analysis ofmeiosis revealed that some of the spindles fused in one or two poles, i.e. fused (bipolar)spindle and tripolar

13、spindle. The results revealed that the abnormal spindles causedirregular operation of microtubules leading to spindle poles fusion. Dyads and triadswere induced by the fusion of spindles and induced unreduced pollen formation. Inaddition, the premature cytokinesis during the second meiotic division

14、also produceddyads. The abnormal spindles produce unreduced pollen is genetically equivalent tofirst-division restitution (FDR) mechanism, while that from premature cytokinesis isgenetically equivalent to second division restitution (SDR).3. Molecular markers of amplified fragment length polymorphis

15、m (AFLP) and SCAR(sequence-characterized amplified region) were employed to identify related molecularmarkers of unreduced pollen in Chinese white poplar. Following an initial screeningwith55&(8HJLTZ?z?z?bd?6t?*蜾巰畔保槑仒帢 x 榵榵榵榵榵榵槑榵榵榵槑 px 榵槑? h6eCJOJ h6eCJOJo( h6e6丆 JOJQJo( h6eCJOJQJ h6eCJOJQJo( h6

16、e h6e5丆 JaJo( h 羥 h 羥5丆 JfHo(q? h 羥 h 羥5丆 J h 羥 h 羥5丆Jo( hFN5丆 Jo( h 羥 hdG? 5丆 J$ aJ$ o( h 羥 h 羥5丆 J$ aJ$ o(, (L?6?gd6e dDH$ gd6e 勦 dhWD? 勦gdFN 勦 dhWD? 勦gd6e? 勦 dhVDWD? ? 勦gd6e dhgdFN $ ? WD? ? a$gd 羥 $a$gd羥0? *0j? x? 鯖鮫限膂膂肱僅僅烹鯖鯖霰獰枱唟 pbV h6e h6e5丆 J$ aJ$ h6e h6e5丆 J$ aJ$ o( h ?P 5丆 JaJo( h6e5丆 JaJo(

17、- h6eCJfHo(q? h6eCJOJo( h6eCJOJQJo( h6eCJo( h6e h6e5丆 Jo( h6eCJOJ h6eCJOJQJ h6eCJOJQJmHo(sH h6e6丆 JOJQJo( h6eCJOJQJo( h6eCJOJo(! T? .000? dDVD?gd6e 勦 dhWD? 勦gdFN 勦 dhWD? 勦gd6e dhgdFN $a$gd6e $ d,a$gd6egd6e Tev? $6Sdyz?Xa?(Rg?#%(Rfz$()-01蜾劁麓牔檺牔牔牔牔牔牔牔牔牔牔牔牔牔牔牔牔 h6e6丆 Jo( h6e6丆 J h6eCJ h6eCJo( hFNCJo( h

18、FN hFN5丆 JaJo(+ h6e h6e5丆 JaJfHo(q? h6e h6e5丆 JaJ h6e h6e5丆 JaJo( h6e h6e5丆 J$ aJ$ o(81?dh? -8:j %)5Wfghm|? *D?,B,C,q,z,? -%-7-8-?-.S./E/T/z/?聱篾篾篾篾篾篾篾篾篾篾篾篾篾篾篾? U h6eCJo( h6eCJ_primer combinations, the E31-M50 (AAA/CAT) primer was identified: it generated a PCRfragment (204 bp) from the unreduced pol

19、len producers, but not from the normal clones.In addition, the E50-M38 (CAT/ACT)-amplified DNA fragment (246 bp) was present in normalclones, and absent in unreduced pollen producers. These two discriminating AFLP markerswere developed into easily detectable SCAR markers which can be used in combina

20、tionwith the previously developed AFLP markers to distinguish between normal and unreducedpollen clones.4. Differential gene expression profiles of meiosis during unreduced pollen formationwere analyzed by cDNA-AFLP technique. From 12 primer combinations, 52 regulateddifferentially transcription-derived fragments (TDFs) wer