【作者】 彭远英；

【导师】 郑有良； 魏育明； Bernard R.Baum；

【作者基本信息】 四川农业大学 ， 植物资源的保护与利用， 2009， 博士

【摘要】 燕麦属(Arena L.)隶属于禾本科(Poaceae),早熟禾亚科(Pooideae),燕麦族(Aveneae),全世界约29个种,含有6种染色体组组成类型(A、C、AB、AC、CC和AACCDD)。燕麦属不同倍性以及不同基因组构成的物种是栽培燕麦产量、品质和抗性改良的重要基因库。但是,相似的形态和重叠的地理分布使燕麦属部分物种的分类与鉴定存在困难;染色体组构成和倍性差异更增加了分类鉴定的复杂性,这种情况阻碍了对这些燕麦物种优异遗传资源的有效利用。此外,由于缺乏明确的二倍体供体物种,燕麦属异源多倍体的起源一直存在争议。本文通过细胞质基因组的扩增片段限制性片段长度多态性(PCR-RFLP)和叶绿体微卫星标记(ccSSR)分析;叶绿体matK基因、trnL内含子和trnL-F基因间隔区、核糖体5S rRNA基因和nrDNA的内转录间隔区(ITS)以及低拷贝的核基因LEAFY的内含子等序列比较分析的方法,对燕麦属种间和基因组间的系统发育关系,以及异源多倍体的起源和ITS的分子进化等进行了研究,并尝试开发燕麦属基因组和物种鉴定的特异标记。主要研究结果如下:1.采用PCR-RFLP技术对燕麦属25个种的95个居群以及1个外类群共96份材料细胞质基因组DNA片段的遗传变异进行了分析。利用12个细胞质通用引物扩增出片段,然后用8种限制性内切酶对扩增产物进行酶切,共获得203条DNA片段,其中仅39条具有多态性,占19.2%,结果表明该标记在燕麦属中多态性很低,不能有效区分不同的基因组、物种或种内居群,因此对于燕麦细胞质基因组的研究需选用多态性更高的标记或直接比较不同物种的叶绿体或线粒体DNA序列。2.利用叶绿体微卫星引物对燕麦25个物种80份材料的SSR位点进行了扩增。16个ccSSR位点检测到51个等位基因,多态信息含量最高为0.754。80份供试材料的平均遗传相似系数为0.545。采用鉴别分析法,评价了ccSSRs分子标记在区分燕麦不同基因组方面的价值。遗传相似性分析结果表明,燕麦属中A基因组二倍体物种相对于其它物种具有更大的遗传分化。根据叶绿体标记母性遗传特点,推测A.damascena可能是A.fatua的母本,而A.strigosa和A.lusitanica分别与不同六倍体物种以及AACC基因组四倍体物种的关系较近,表明多倍体燕麦物种的A基因组是从不同AA基因组的二倍体物种起源的。C基因组物种与其它基因组类型的物种能明显区分,但C基因组二倍体A.clauda有分布在不同组群内的个体,可能该物种在燕麦进化过程中具有比较重要的作用。3.根据叶绿体基因组母系遗传特点,选择matK和trnL-F两个叶绿体基因片段探讨燕麦属异源多倍体物种的母系亲本。在本研究中,代表A基因组的分支包含了燕麦属中含A基因组的所有倍性的物种,而且在两个不同的片段独立和合并的分析中这些物种都以很高的自展支持率聚为一支,说明A基因组二倍体作为燕麦多倍体物种的母本参与燕麦属多倍体物种的形成,且不同多倍体物种具有的A基因组二倍体祖先也不同。六倍体物种A.sativa、A.sterilis和A.occidentalis,AC基因组四倍体物种A.maroccana和A.murphy,以及AB基因组四倍体物种A.agadiriana一起与二倍体物种A.wiestii组成一支,A_d基因组二倍体物种A.damascena与六倍体物种A.fatua的遗传关系更近;A.hirtula可能是A.abyssinica、A.vaviloviana和A.barbata等三个AABB物种的母本。因此,燕麦属多倍体物种具有多系母本起源,而并非都起源于同一个母本。4.鉴于ITS片段进化的特殊性以及该片段一直被作为致同进化的经典范例而广泛应用于系统发育研究,基于169条ITS序列的比较,探讨其在燕麦属多倍体中的致同进化方式及其系统学意义。本研究发现,燕麦属异源多倍体的ITS存在不同进化方式。首先,ITS定向致同进化方式在大部分燕麦异源多倍体中出现。在AABB和AACC四倍体中,所有ITS克隆序列都毫无例外地出现在含A染色体组的二倍体所在的分支中,没有其它类型的拷贝出现。在5个燕麦属六倍体物种中,有4个物种的ITS序列均属于A基因组的ITS类型。这些结果表明燕麦属大多数多倍体的rDNA仅定向保留母本的ITS类型。其次,在六倍体物种A.fatua(AACCDD)中同时保留了两类分别来自亲本AA和CC的ITS拷贝类型。因此,在利用ITS片段进行系统发育分析,特别是涉及异源多倍体时必须考虑到该片段特殊的进化模式,以避免作出错误的系统发育推断。5.对从燕麦属26个物种71个居群材料中获得的553条5S rDNA序列进行比较分析,发现可将所有序列划分为代表燕麦各基因组的六种单元类型,分别命名为Long A1、Long B1、Long M1、Short C1、Short D1和Short M1,而Long M1和Short M1仅在燕麦属唯一的多年生物种A.macrostachya中发现。其中,Long M1单元类型与代表C基因组的Short C1单元类型更相似,Short M1单元类型则与Long A1和Long B1关系更近,而Short D1单元类型与其它类型差异较大。与小麦不同,燕麦属每个基因组仅发现一种5S rDNA单元类,且大多数序列都属于Long A1类型。从四倍体A.abyssinica和A.vaviloviana以及二倍A.atlantica和A.longiglumis中发现了代表B基因组的Long B1单元类型。Short C1单元类型在C基因组二倍A.clauda、A.eriantha和A.ventricosa中发现,同时还在二倍体A.longiglumis、四倍体A.insularis和A.maroccana以及所有的ACD基因组六倍体物种中存在。Short D1单元类型也存在于所有的六倍体物种中。同时,在C基因组二倍体物种A.clauda和A.murphyi冲也发现了代表D基因组的拷贝类型。虽然B基因组和D基因组都仅在多倍体中存在,但本研究从二倍体物种中发现了代表B基因组和D基因组的单元类型。这就为今后寻找多倍体中B基因组和D基因组的起源提供了线索。在燕麦A、B、C、D四个基因组中,燕麦属最原始的多年生物种A.macrostachya与C基因组关系更近。6.利用具有更多系统进化信息位点的低拷贝核基因片段LEAFY intⅡ对燕麦属内种间进化关系和多倍体起源进行研究,找到了B基因组和D基因组起源的线索。三个AB基因组四倍体物种A.abyssinica、A.barbata和A.vaviloviana的B基因组可能由二倍体物种A.hirtula起源,而另一个AABB物种A.agadiriana的B基因组则可能起源于二倍体物种A.damascena。对于仅在六倍体中存在的D基因组则可能是起源于含有D类拷贝的C基因组二倍体物种A.clauda和A.eriantha,以及四倍体物种A.murphyi。同时,燕麦属多倍体物种中的C基因组显示了与C_p基因组物种A.clauda更近的遗传关系。7.根据5S rRNA基因序列在不同基因组间的差异设计基因组特异引物对燕麦属物种进行扩增,探索了全面获取不同物种所有序列拷贝类型和鉴别燕麦属染色体组的方法。已找到能准确鉴定A基因组和D基因组的特异引物,显示出开发燕麦属基因组特异标记的可行性和应用价值。同时,这些特异引物还可用于燕麦种质资源的快速准确鉴定。更多还原

【Abstract】 The genus Avena L. in the tribe Aveneae, subfamily Pooideae, Poaceae, consists of 29 species, including 6 genome constitutions (A, C, AB, AC, CC, AACCDD). As an important gene pool for improving cultivated oats and related species, this genus occupies a most important status in cereal plants. However, the species in this genus are often overlapping in ditribution and morphologically so similar that it is very difficult to identify them with certainy if only using gross-morphological charecters. Difference in genome constitution and ploidy level add even greater complexity. Such situation has undoubtedly restricted the effective utilization of the the valuable genetic resources in the genus. In additon, the incomplete knowledge on the diploid donors of the polyploid members in the genus has made the situation become more complicated. Indeed, the origin of the allopolyploid members in the genus has long been a controversial matter. In this study, based on PCR-RFLP and ccSSR analysis of plasmon, the plastid matK gene and the trnL-F region, the nuclear ribosomal internal transcribed spacers (ITS) and 5S rRNA gene, and the intron region of low-copy LEAFY gene, the phylogenetic relationships of the species and the origin of the allotetraploid members were discussed. In additon, based on the former results, primers specific for genome were designed and used to identify the different genomes. The main results are summarized as follows:1. Plasmon genetic varitions of 96 accessions including 25 Avena taxa and 1 outgroup were investigated by using PCR-RFLP markers. Twelve plasmon universal primers produced 203 bands, only 39 out of which were polymorphic (19.2%), indicating that it is not suitable for identify the different genome, species or accessions of Avena. Thus, the consensus chloroplast simple sequence repeat (ccSSR) makers and plastid matK gene and the trnL-F region were used to study the genetic polymorphisms of the Avena plasmon.2. Consensus chloroplast simple sequence repeat (ccSSR) makers were used to assess the genetic variation and genetic relationships of 80 accessions from 25 taxa of the genus Avena. A total of 51 alleles were detected at the 16 ccSSR loci. Among these ccSSR loci, the highest polymorphism information content (PIC) value was 0.754. The mean genetic similarity index among the 80 Avena accessions was 0.545. To assess the usefulness of ccSSRs in separating and distinguishing between haplome (genome) groups, we used ordination by canonical discriminant analysis and classificatory discriminant analysis. The analysis of genetic similarity showed that diploid species with the A haplome were more diverse than other species. Among the species with the C haplome, A. clauda was more diverse than A. eriantha and A. ventricosa. In the cluster analysis, we found that the Avena accessions with the same genomes and/or belonging to the same species had the tendency to cluster together. As for the maternal donors of polyploid species based on this maternally inherited marker, A. strigosa served as the maternal donor of some Avena polyploidy species such as A. sativa, A. sterilis and A. occidentalis from Morocco. A. fatua is genetically distinct from other hexaploid Avena species, and A. damascena might be the A genome donor of A. fatua. A. lusitanica served as the maternal parents during the polyploid formation of the AACC tetraploids and some AACCDD hexaploids. These results suggested that different diploid species were the putative A haplome donors of the tetraploid and hexaploid species. The C genome species A. eriantha and A. ventricosa are largely differentiated from the Avena species containing the A, or B, or D haplomes, whereas A. clauda from different accessions were found to be scattered within different groups, and might play an important role in the phylogeny of Avena.3. The maternally inherited matK and trnL-F sequences revealed the separate A genome diploid species as maternal parents of the different polyploid species. One of the A_SA_S donor diploid species, A. wiestii, fell together with most hexaploid species, A. sativa, A. sterilis and A. occidentalis, with the two AACC tetraploids, A. maroccana and A. murphy, and with one of the AABB genome tetraploid, A. agadiriana on the chloroplast gene trees. Another diploid species, A. damascena carrying the A_dA_d genome, always fell together with the hexaploid A. fatua. Three AABB tetraploids, A. abyssinica, A. vaviloviana and A. barbata, were close to the AA genome diploid A. hirtula in a subclade on the tree. Therefore, the maternal donor was an A genome species with several maternal lineages being involved in different polyploid species.4. ITS concerted evolution in the genus of Avena was detected based on the comparison of 169 clone sequences, and phylogenetic implication of such evolution was discussed. Different patterns of concerted evolution are revealed in the allopolyploid members if the genus. First, only maternal ITS copies are found to be reversed within the most hexaploids and all of the AABB and AACC tetraploids, indicating the high degree of homogenization in the ITS sequences of Avena. Second, biparental ITS copies are both found within the A. fatua, which had two types in separate clades, one with A genome and the other with C genome species. It need more consideration when utilizing ITS for phylogenetic reconstruction.5. The molecular diversity of the rDNA sequences (5S rDNA units) in 71 accessions from 26 taxa of Avena was evaluated. The analyses based on 553 sequenced clones, indicated that there were six sequence unit classes, enumerated according to the haplomes (genomes) they putatively represent, namely the Long A1, Long B1, Long M1, Short C1, Short D1 and Short M1 unit classes. The long and short M1 were found in the tetraploid A. macrostachya the only perennial species. The long M1 unit class was closely related to the short C1 unit class, while the short Ml unit class was closely related to the long A1 and the long B1 unit classes. However, the Short D1 unit class was more divergent with the other unit classes. There was only one unit class per haplome in Avena, which was different from haplomes in the Triticeae which often have two haplomes. Most of the sequences captured belong to the long A1 unit class. Sequences identified as the long B1 unit class were found in the tetraploids A. abyssinica and A. vaviloviana, and in the diploids A. atlantica and A. longiglumis. The short C1 unit class was found in the diploid species carrying the C genome i.e. A. clauda, A. eriantha and A. ventricosa, and also found in the diploid A. longiglumis and in the tetraploid A. insularis, A. maroccana and in all the hexaploid species. The short D1 unit class was found in all the hexaploid species and in two clones of A. clauda and A. murphyi. It is noteworthy that, according to previous studies the B genome was found to be present only in tetraploid species and the D genome only in hexaploid species. Unexpectedly we found that various diploid Avena species contained the B1 and D1 units. Thus there might be a clue where to search for the diploids carrying the B and D genomes. The path inferred is that the C genome is more ancient than the A and B genomes, and it is closer to A. macrostachya, the only existing perennial which is presumed to be the most ancestral species in the genus.6. The usefulness of low-copy nuclear sequence markers is becoming increasingly recognised since they frequently outperform ITS and plastid markers, the highly variable second intron region of low-copy gene LEAFY was used to inferring the phylogenetic relationships of Avena species and the origin of the allopolyploids, especially for the orign of the B and D genome which only found in polyploids. The B genome of three AABB tetraploids, A. abyssinica, A. barbata and A. vaviloviana, were from the AA genome diploid A. hirtula, and another B genome copy from A. agadiriana was closed to the AA genome diploid A. damascena. As for the D genome which only present in hexaploids, it maybe originate from CC genome diploids A. clauda and A. eriantha, or from the tetraploid A. murphyi which contained the D genome copy. The C genome of polyploids showed closer relationship with one of the CC genome species A. clauda.7. It is an opporunity to design primers specific for genome based on the 5S rDNA class units which different genomes has specific sequence varations. The specific PCR primers had been developed to identify A and D genome of Avena species, indicating the feasibility and value of the genome specific. According to the phylogenetic study of the Avena genus, the amount of sequence variation in the 5S rRNA gene and LEAFY second intron region seems to be sufficient to allow the development of genome specific primers or RFLP markers for the identification of the Avena genomes. It is necessary to explore more genome specific molecular markers in future in order to develop an approach of PCR or RFLP analysis to identify the Avena genomes quickly and reliably. This can be used to affirm whether all copy types of certain species have been obtained and would greatly facilitate the identification of collected oat germplasm.更多还原