【作者】 崔海峰；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 博士

【摘要】 体细胞杂交技术能够克服有性杂交不亲和等障碍,产生多种多样的有性杂交无法实现的作物新类型。目前,体细胞杂交育种已在多种植物中获得成功。本实验室成功的进行了小麦与多种禾草的体细胞杂交,并在小麦与长穗偃麦草的对称与不对称体细胞杂交中分别获得了长穗偃麦草/小麦渐渗系和小麦/长穗偃麦草渐渗系。通过对小麦/长穗偃麦草渐渗系的筛选培养,从其后代中选育出一系列耐盐、耐旱新品种和高产、优质的小麦新品系。对小麦体细胞杂交机制进行深入研究将有助于体细胞杂交技术在小麦育种中的应用,但是目前尚无相关报道。本实验对小麦体细胞杂交早期供体染色质消减与渐渗机制进行了系统的研究,阐明了小麦体细胞杂种中供体染色质消减的时期及方式,探讨了供体与小麦亲缘关系及射线剂量对小麦体细胞杂种中供体染色质消减和渐渗的影响;通过混合小麦与两种不同亲缘关系供体的对称体细胞杂交,首次获得了小麦与早熟禾的体细胞杂种,揭示了小麦杂种的植株再生与亲本染色质互补现象,并探讨了组织培养及杂交过程中的甲基化模式变化。此外,通过对小麦与长穗偃麦草的偏草型杂种及后代的分析,阐明了杂种小麦染色体的存在方式;探讨了杂种中小麦染色体的消减现象。主要研究结果如下:1小麦体细胞杂交早期杂种细胞中供体染色质的消减方式小麦与四种禾草(长穗偃麦草Agropyron elongatum(Host)Neviski、簇毛麦Haynaldia villosa L.、早熟禾Poa annua L.、玉米Zea mays L.)体细胞杂交早期融合材料的GISH分析表明:杂种中供体染色质主要以微核的形式在细胞间期发生消减,同时还存在少量其它的消减方式,如有丝分裂后期形成微核、多核体中形成微核以及双亲核分离等现象。此外,在簇毛麦的体细胞杂种中发现供体染色质以落后染色体及染色体桥的形式存在。分析发现双亲核分离的现象主要出现在小麦与远缘禾草早熟禾和玉米的杂交组合5-7天的材料中,而在近缘禾草与小麦的杂交中很少出现,表明亲缘关系对小麦体细胞杂交中供体染色质的消减具有明显的影响。2小麦体细胞杂交早期杂种细胞中供体染色质消减与渐渗发生的主要时期小麦与簇毛麦及早熟禾γ-ray不对称体细胞杂交分析发现:杂交后5—7天供体染色质的消减率与渐渗率明显高于杂交后30天;融合后30天供体染色质的消减率与渐渗率均低于10%。由此可见,小麦体细胞杂交过程中供体染色质消减与渐渗主要发生在杂交后30天以内,尤其是杂交后5—7天。此外,在小麦与四种禾草的对称与UV不对称体细胞杂交中,杂交后5—7天供体染色质的渐渗率与消减率均明显高于12—15天,这进一步表明小麦体细胞杂交中供体染色质的消减与渐渗主要发生在杂交早期。3供体与小麦的亲缘关系对小麦体细胞杂交早期杂种细胞中供体染色质消减与渐渗的影响通过对小麦与近缘禾草长穗偃麦草、簇毛麦以及远缘禾草早熟禾、玉米的对称与不对称体细胞杂交(UV 1min和UV 2min)的比较分析发现,供体与小麦的亲缘关系对供体染色质的渐渗率具有非常明显的影响。亲缘关系近,则杂种中供体染色质的渐渗率高;亲缘关系远,则杂种中供体染色质的渐渗率低。此外,供体与小麦的亲缘关系与杂种中供体染色质的消减率的变化也具有一定的规律性:亲缘关系近,则杂种中供体染色质的消减率低;亲缘关系远,则杂种中供体染色质的消减率高。但是在长穗偃麦草的组合中供体染色质的消减率均很高,如UV1min的剂量组合中,5—7天的消减率为50.4%,12—15天的为37.2%,明显高于簇毛麦的消减率(37.0%和22.7%)。这表明:近缘供体的倍性及染色体组的大小也可以影响小麦体细胞杂交中供体染色质的消减率。4射线剂量对小麦体细胞杂交早期杂种细胞中供体染色质的消减与渐渗的影响通过对小麦与四种禾草不对称体细胞杂交中供体染色质消减与渐渗的比较分析表明:小麦体细胞杂交中供体染色质的消减与渐渗受射线剂量的影响。在一定的剂量范围内,供体染色质的渐渗率随着射线剂量的增加而升高,供体染色质的消减率随射线剂量的增加而降低。当射线剂量超出一定范围时,如早熟禾γ-ray不对称杂交10000rad剂量组合中,随着射线剂量的增加,供体染色质的渐渗率明显降低,而消减率大幅升高。5供体与小麦的亲缘关系对混合小麦体细胞杂种不对称程度的影响混合小麦(cha9和176)与簇毛麦及早熟禾的对称体细胞杂交获得了不对称程度不同的体细胞杂种,并再生大量杂种植株。对两个组合体细胞杂种的GISH分析表明:小麦/簇毛麦体细胞杂种中,簇毛麦染色体主要以整条的形式存在,同时有1-2个簇毛麦染色体片段;而小麦/早熟禾的体细胞杂种中,没有发现早熟禾的整条染色体,其染色质呈点状散布在杂种染色体上。小麦/早熟禾体细胞杂种的不对称性远远高于小麦/簇毛麦体细胞杂种。由此可见,供体与小麦亲缘关系越远,小麦体细胞杂种的不对称程度越高。6体细胞杂种再生与亲本染色质的互补以含有18S-5.8S-26SrDNA特异序列的pTa71质粒为探针,对同一基因型的愈伤组织(济南177)和悬浮细胞系(cha 9)混合小麦亲本及体细胞杂种进行了FISH分析。首次发现了混合小麦中不同的NORs区:cha9保留了亲本小麦中5条染色体上的5个信号,而176仅保留了2条染色体上的2个信号;cha9和176各自含有小麦济南177所没有的特异NORs区信号。在早熟禾杂种16H中检测到混合小麦各自特异的NORs区信号,直观的揭示了混合小麦在杂种染色体组成上的互补现象。通过对杂种NORs区信号及杂种表型的分析表明杂种存在明显的混合小麦再生互补现象。核基因组SSR分析也证实了这一点。混合小麦及早熟禾体细胞杂种4H的MSAP分析发现,经过长期的继代培养,混合小麦发生了大量的甲基化模式改变,有大量位点发生甲基化。杂种植株再生过程中也有甲基化模式的改变,但明显低于混合小麦。表明甲基化模式变化与组织培养的时间有关。杂种4H愈伤组织与cha9和176相比,甲基化模式变化明显不同。与小麦177相比,杂种植株再生时,体细胞杂种中甲基化模式变化得到了明显的恢复,包括5.88%的甲基化位点和3.35%的去甲基化位点,表明体细胞杂交中的再生互补与表观遗传变化相关。7对称体细胞杂交中小麦染色体的消减与渐渗染色体分析发现小麦与长穗偃麦草偏草型体细胞杂种染色体数目与长穗偃麦草类似,对R0粗杂种植株部分PMC的GISH及FISH分析发现:小麦染色体在减数分裂末期Ⅰ时,明显滞后,停留在赤道板上,与长穗偃麦草分离:R2代PMC中仅检测到3-5个小麦染色体小片段。这表明小麦染色体在杂种进入有性世代或减数分裂过程中大量消减,并在消减的过程中与长穗偃麦草染色体发生重组,最终以染色体小片段的方式渐渗进杂种中。对小麦与长穗偃麦草偏草型体细胞杂种的AFLP分析发现,在R2代杂种中,绝大多数小麦的基因组发生消减,在两个杂种中仅检测到13条小麦特异条带,其中9条在杂种中共存,1条仅在粗系杂种存在,其余3条仅在细系杂种中存在;两个杂种中长穗偃麦草基因组消减频率分别为9.8%及13.4%。此外,在两个杂种中还检测到19条新带,其中三条仅在细系杂种中出现。这表明体细胞杂种中双亲基因组均发生了消减,同时伴随着基因组的重排。对双亲及R1-R3代部分杂种种子进行的HMW-GS分析发现,杂种后代的HMW-GS带型主要偏向长穗偃麦草,杂种中有三个长穗偃麦草的HMW-GS丢失,同时有三个新的HMW-GS出现,表明体细胞杂种的HMW-GS发生了明显变化。更多还原

【Abstract】 Somatic hybridization has been suggested as a means of circumventing interspecific barriers to fertilization and has been successfully used in plant breeding. In our lab,studies on the somatic hybridization between wheat and many other genera of grasses have been processed for a long time,and somatic hybrid plants and their offsprings have been obtained from some combinations.When asymmetric somatic hybridization was applied to the bread wheat / tall wheatgrass combination,a number of high yielding and good quality introgression lines,carrying resistance to stripe rust, tolerance to salinity and drought have been generated.Besides,two fertile hybrids were obtained from symmetric somatic hybridization between bread wheat and tall wheatgrass,both of which were phenotypically similar to the tall wheatgrass parent. Although it is important for crop breeding,the mechanism of somatic hybridization is still unclear.Systemic investigation about chromatin elimination and introgression of donor in wheat somatic hybridization were progressed in this research.Time and fashion of chromatin elimination and introgression of donor were discovered. Influence of phylogenetic relationship of wheat with donor and radiation dose on chromatin elimination and introgression of donor were discussed.Besides,through symmetric somatic hybridization between wheat and two grasses（Poa annua L and Haynaldia villosa L）,we investigated the complement of two types of calli of wheat in hybrids and the change of methylation in culture and somatic hybridization.Finally, somatic hybrids between wheat and tall wheatgrass were analyzed,which phenotype was tall wheatgrass-like.Chromosome elimination and introgression of wheat in hybrids were discovered.The sequence elimination of parents and change of HWM-GS in hybrids were also discovered.The main results of this research are listed as follows: 1.Fashions of chromatin elimination of donor in early stage of wheat somatic hybridizationAfter GISH analysis of somatic hybridization between wheat and four different donors（A.elongatum,H.villosa,P annua,Z.mays）,we found that donor chromatin were mainly eliminated in interphase through micronucleus formation in early stage of wheat somatic hybridization.There were also other fashions of chromatin elimination of donor in early stage of wheat somatic hybridization,including micronucleus formation in anaphase,micronucleus formation in multi-nucleus and separation of parental nucleus.Besides,we found lagging chromosomes and chromosome bridges in somatic hybridization between wheat and H.villosa. Separation of parental nucleus were mainly occurred in remote somatic hybridization between wheat and donor,especially in the time of 5-7d after fusion.2.Chromatin elimination and introgression of donor in early stage of wheat somatic hybridizationAfter analysis of asymmetric somatic hybridization usingγ-ray between wheat and two grasses（P.annua and H.villosa）,we found that the introgression frequency and elimination frequency of donor after fusion were all clearly higher in 5-7d than in 30d.The introgression frequency and elimination frequency of donor were lower than 10%in 30d.Besides,we analyzed asymmetric somatic hybridization using UV between wheat and four donors.The introgression frequency and elimination frequency of donor after fusion were also clearly higher in 5-7d than those in 12-15d. In conclusion,chromatin elimination and introgression of donor in early stage of wheat somatic hybridization were mainly occurred in 5-7d after fusion.3.Influence of phylogenetic relationship on chromatin elimination and introgression of donor in early stage of wheat somatic hybridization After analysis of somatic hybridization between wheat and four donors,which were different from wheat in phylogenetic relationship.We found that the introgression frequency of donor was clearly disparate in different somatic hybridization combinations between wheat and distinct donors.Compared to the combinations of P annua and Z.mays,which were remote to wheat,the introgression frequency was higher in the combinations of tall wheatgrass and H.villosa which were close to wheat.But elimination frequency of donor was lower in the combinations of H.villosa than that in the combinations of P annua,and Z.mays. Because of the larger polyploid of A.elongatum than H.villosa,the elimination frequency of donor in combinations of tall wheatgrass was higher than that in combinations of H.villosa.It was clear that chromatin elimination and introgression of donor in early stage of wheat somatic hybridization could be affected by the relationship of wheat and donor.When the donors had related relationship to wheat, the polyploidy of donor may also affect chromatin elimination and introgression of donor in early stage of wheat somatic hybridization.4.Influence of radiation dose on chromatin elimination and introgression of donor in early stage of wheat somatic hybridization Compared analysis of asymmetric somatic hybridization between wheat and four grasses,we found that radiation dose was related to chromatin elimination and introgression of donor in early stage of wheat somatic hybridization.Under a certain range of radiation dose,introgression of donor increased following the increasing radiation dosage,while chromatin elimination of donor decreased following the increasing radiation dosage.When radiation dose was higher than the range, elimination of donor increased abnormally following the increasing radiation dose.5.Effect of different relationship between wheat and donor on asymmetric degree of hybrids in symmetric somatic hybridization Through symmetric somatic hybridization between P.annua and two different wheat callus（cha9 and 176） which were captured from the same wheat（Jinan177）,we get wheat / P.annua asymmetric somatic hybrids and hybrid plants.Compared with somatic hybrids of wheat with H.villosa,spot chromatin of P.annua were diffused in wheat chromosomes in wheat / P.annua hybrids,while intact chromosomes or chromosome segments of H.villosa were existed in wheat / H.villosa hybrids.The asymmetric degree of wheat / P.annua somatic hybrids was much higher than wheat/ H.villosa somatic hybrids.It was clear that the remote donor could get high asymmetric hybrids in wheat symmetric somatic hybridization. 6.Regeneration of somatic hybrids with complementation of parental chromatinUsing the special NORs sequence of 185-5.85-265rDNA as probe,FISH analysis was processed in the parents and somatic hybrids.We found that the distribution of NORs were different in the two wheat callus.Five chromosomes of Jinan 177 which could detect the NORs signals were detected in cha9,while just two chromosomes of Jinan 177 were detected in 176.Besides,special signals of NORs were detected in cha9 and 176 separately,which were caused by culture.These special signals were all detected in a wheat / P annua somatic hybrids（16H）,and directly proved the complement of cha9 and 176 in wheat somatic hybrids.The phenotype and SSR analysis of hybrids also illuminated this complement.MSAP analysis of wheat callus and wheat / P annua somatic hybrids（4H） showed that there were clear changes of methylation.Compared with Jinan 177,there were many changes of methylation in cha9 and 176,which were caused by culture. Besides,there were also changes of methylation in hybrids between callus and plants. Both of these showed that changes of methylation were related to the length of culture time.Compared with Jinan 177,callus and plants of somatic hybrids had different changes of methylation.Some changes of methylation in callus were recovered, including 5.88%of methylation and 3.35%of demethylation.7.Chromosomes elimination and introgression of wheat in symmetric somatic hybridsChromosome analysis of the wheat/tall wheatgrass somatic hybrids showed that the number of hybrids were similar to tall wheatgrass（Agropyron elongatum（Host） Neviski）.Some lagging chromosomes at telophaseⅠwere observed,as chromosome bridges at anaphaseⅠand micronuclei at telophaseⅡ.After GISH analysis of the PMC of R0 hybrids,we found the existence of wheat genomes and the asynchronism division in some hybrid cells of R0 plants,In the meiosis of some R₂ individuals,GISH was able to characterize the inheritance of introgressions in the hybrid genome.This analysis showed that all complete wheat chromosomes were eliminated,but that segments were introgressed into tall wheatgrass chromosomes.Of the 745 fragments scored in AFLP analysis,305 were specific to tall wheatgrass,and 267 to wheat.XI and CU carried a different set of wheat-specific and tall wheatgrass-specific fragments.They shared 256 tall wheatgrass-specific fragments but differed with respect to 27 fragments,of which 19 were carried by CU and eight by XI.Of the 13 wheat-specific fragments present in the R₂ lines,nine were present in both CU and XI,three was only present in XI and one in CU.The 13 wheat-specific fragments were detected by six of the 13 primer combinations.There were also 17 novel bands in each R₂ hybrids and another two were only present in XI.All of these showed that both wheat and tall wheatgrass sequences were lost or recombined.The HMW-GS profile of direct and later generation progeny of the two R₂ lines differed from that of tall wheatgrass by the absence of three subunits,which were replaced by three subunits not present in either the tall wheatgrass or the bread wheat parent.更多还原