【作者】 陈其皎；

【导师】 刘登才；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 在自然界,由不同物种杂交后经染色体组加倍产生的异源多倍体植物非常普遍。普通小麦（Triticum aestivum L.,染色体组为AABBDD,2n=6x=42）是异源多倍体物种的一个典型代表,它是由栽培四倍体小麦为母本与节节麦（Aegilops tauschii）为父本天然杂交,然后通过染色体自然加倍形成的新兴异源六倍体物种。模拟小麦起源过程可以合成新六倍体小麦（人工合成小麦）。近年围绕人工合成小麦进行的相关研究,已为异源多倍化起源机制探讨和异源多倍体作物种质资源开发及遗传育种研究提供了十分重要的参考信息。本研究以新合成的人工小麦群体为材料,对微卫星（SSR）产物进行了详细的比较研究,主要研究结果如下:利用四倍体硬粒小麦（T.turgidum L.ssp.durum）Langdon（LDN）与节节麦AS60远缘杂交,不使用幼胚培养处理获得的4个F₁杂种自交分别自发产生了20,19,23,19株具有42条染色体的F₂植株,构建了遗传研究群体。运用108对核基因组SSR引物,对4个F₁杂种、81个F₂株系及其亲本的SSR产物进行比较研究发现:（1）有105对引物得到可以辨别的扩增产物。其中,55引物对来自普通小麦或节节麦的D-基因组,46对分别来自普通小麦的A或B基因组,1对来自普通小麦的A/B/D基因组,1对来自普通小麦的A/D基因组,2对来自普通小麦的B/D基因组。在55对D-基因组引物中,18对从LDN扩增出SSR产物,表现出可转移性或非D基因组特异性,占18/55=32.7%;但是,在46对A/B基因组引物中,仅6对从Ae.tauschii AS60的D基因组扩增出SSR产物,表现出A/B基因组非特异性的占6/46=13.04%,大大低于前者;（2）105对SSR引物中,9对引物的SSR产物在合成小麦群体中发生了变异,占9/105=8.57%。其中,在55对D-基因组引物中,5对揭示了变异（5/55=9.09%）,包括2对引物在四倍体小麦LDN的SSR片段在合成小麦中丢失,2对引物在合成小麦中产生新片段（2/55=3.64%）,1对引物在部分F₁植株中的SSR产物消失,但在其F₂群体中又出现;在46对A/B基因组引物中,3对揭示了变异（3/46=6.52%）,包括2对引物在合成小麦中产生新片段,1对引物在合成F₂群体中的一些植株带纹变小。D基因组SSR的变异高于A/B基因组的SSR变异。此外,1对A/D基因组引物从Ae.tauschii扩增的产物在部分合成的F₂植株中消失;（3）杂交世代和加倍世代都可能引起SSR变异,这样的变异可能导致新六倍体小麦群体的SSR遗传多样性增加。但是从本研究结果看,杂交世代和加倍世代引起SSR变异的频率并不高。相反,现存普通小麦存在非常高的SSR遗传多样性。根据本研究结果,讨论了现存普通小麦大量SSR遗传变异的产生等问题。运用24对叶绿体基因组SSR引物,对上述杂种4个F₁、81个F₂及其亲本的SSR产物进行的比较,未观察到SSR大小上的差异、新带出现或亲本带纹丢失等变异。我们对其中的4对SSR引物的扩增产物进行测序,却发现有2对引物在F₁代的部分植株发生了碱基突变,而且该突变传递到其F₂代植株。这表明,细胞质基因组在合成小麦后代出现了变异。因为人工合成六倍体小麦与栽培普通小麦之间存在高的多态性,所以人工合成六倍体小麦已被广泛用于遗传研究分离群体的构建。许多从普通小麦开发的特异SSR分子标记可以从供体四倍体小麦和节节麦及它们的人工合成小麦中扩增出SSR产物,表现出可转移性,这暗示对普通小麦来说是单一位点的SSR产物,在人工合成六倍体小麦可能在两个或两个以上位点存在SSR产物,表现出非特异性。在用人工合成小麦群体和SSR进行遗传分析时,这是一个容易被忽略的重要问题。但是,目前还缺乏更为有效的遗传学证据证明这种“非特异性”。本研究利用SSR标记GWM261作为一个例子,详细探讨了该问题。GWM261在普通小麦中国春扩增出了一个192 bp片段,该片段只存在于中国春的2D染色体上。该标记在具有D基因组的节节麦AS60上扩增出了一个176 bp片段。但是,染色体定位和DNA测序表明一个完全相同的176 bp片段也存在于四倍体硬粒小麦Langdon 2B染色体上。用Langdon和AS60合成的人工小麦Syn-SAU-5也出现176 bp片段。推测Syn-SAU-5的176 bp片段包含两个不同的位点,其中一个来自AS60的2D染色体,另一个来自Langdon 2B染色体上,这一推测通过Syn-SAU-5和中国春杂交产生的185个F₂分离群体得到证实。根据上述结果,如果遗传分析中把Syn-SAU-5上的Xgwm261看成一个基因位点,结果就会产生偏分离或得出不正确的结论。为避免该问题,在用人工合成小麦进行SSR多态性检测时,应将其供体四倍体小麦和节节麦也包括进来,如果四倍体小麦和节节麦不存在多态性,则这样的标记不要被采用。更多还原

【Abstract】 Allopolyploid,derived from the duplication of diverged genomes with homoeologous relationships,has been found to be very common in plants. Common wheat（Triticum aestivum L.,2n=6x=42,genome AABBDD）is a good example of allopolyploid,which was formed by the intercrossing between T. turgidum（2n=4x=28,genome AABB）and Ae.tauschii（2n=2x=14,genome DD） followed by chromosome doubling.Common wheat has been an interesting model for the study of the organization and evolution of plant genomes.By the mimic of common wheat origination,many synthetic hexaploid wheats have been produced.In this study,we analyzed microsatellite（SSR）products of newly synthetic wheat populations.The results were as follows:Four F₁ hybrid plants were obtained from the cross of T.turgidum L.ssp. durum cv.Langdon（LDN）with Ae.tauschii accession AS60 without embryo rescue and hormone treatment.By selfing,20,19,23 and 19 F₂ plants with 42 chromosomes were spontaneously produced from the four F₁ plants,respectively. By comparison of nuclear SSR products among the four F₁,81 F₂ plants and their parents LDN and AS60,it was indicated that:（1）105 SSR primers can reveal useful information for further analysis,including in 55 developed from D-genome of common wheat or Ae.tauschii,46 from A or B genome,one from A/B/D genome and two from B/D genome of common wheat.Among the 55 primers developed from D-genome,18（18/55=32.7%）showed transferability and amplified SSR products from A or B genome in LDN;however,a lower transferability was shown in the 46 primers developed from A or B genome. Among the 46 primers,only 6（6/46=13.04%）amplified SSR products from D-genome of Ae.tauschii AS60;（2）most SSR markers were conserved during polyploidization events of newly synthetic wheat.Among 105 primers,only 9 （9/105=8.57%）revealed variations.Of which,five（5/55=9.09%）out of 55 primers developed from D-genome revealed variations.Two primers amplified products from LDN,which were disappeared in newly synthetic wheat.Extra products amplified by two primers were found in newly synthetic wheat. Amplified products from one primer that had disappeared in some F₁s appeared in their F₂ plants.Three（3/46=6.52%）out of 46 primers developed from A or B genome revealed variations.Extra products amplified by two primers were found in newly synthetic wheat.A smaller amplified fragment from one primer was appeared in some F₂ plants.It seemed that the variations revealed by D-genome primers were higher than A or B genome primers.In addition, amplified products from parents by one A/D primer,which were disappeared in F₂ plants;（3）SSR variations can happen in F₁ or F₂,which can lead to a higher polymorphism in newly synthetic wheat population.However,the low variation caused by the allopolyploidization process is not agreed with the very high polymorphism existed in current common wheat cultivars.Based on present results,the origin of high polymorphism in current common wheat was discussed.We also compared SSR patterns revealed by 24 pairs of primers from chloroplast among the four F₁,81 F₂ plants and their parents LDN and AS60. However,there were no variations in SSR patterns.Furthermore,we sequenced and then analyzed the SSR products amplified by four primers.Single base mutation from WCT4 and WCT23 was respectively found in some F₁ and corresponding F₂ plants,thus indicating that varations happened in the LDN×AS60 hybrid process.Due to the high polymorphisms between synthetic hexaploid wheat（SHW） and common wheat,SHW has been widely used in genetic studies.The transferability of SSRs among common wheat and its donor species,Triticum turgidum and Aegilops tauschii,and their SHW suggested the possibility that some SSRs,specific for a single locus in common wheat,will appear in two or more loci in SHWs.This is an important genetic issue when using synthetic hexaploid wheat population and SSR for mapping,however,it is largely ignored and not well verified empirically before.The present study addressed this issue by using the well-studied SSR marker GWM261 as an example.The GWM261 produced a 192 bp fragment specific to chromosome 2D in common wheat Chinese Spring,but generated a 176 bp fragment in the D genome of Ae. tauschii AS60.Chromosomal location and DNA sequence data revealed that the176 bp fragment was also donated by 2B chromosome of durum wheat Langdon.These results indicated that although synthetic hexaploid wheat Syn-SAU-5 between Langdon and AS60 appeared a single 176 bp fragment,the fragment contained two different loci,one from chromosome 2D of AS60 and the other from 2B of Langdon,and were further confirmed by the segregating analysis of SSR GWM261 in 185 plants from a F₂ population between Syn-SAU-5 and Chinese Spring.If GWM261 in Syn-SAU-5 was considered as single locus in genetic analysis,distorted segregation or incorrect conclusions would be yielded.A proposed strategy to avoid this problem is to include SHW’s parental T.turgidum and Ae.tauschii in SSR analysis as control for polymorphism detection.更多还原