【作者】 曾建；

【导师】 周永红； George Fedak； Wenguang Cao；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 小麦族(Triticeae Dumortier)是禾本科(Poaceae)植物中具有最重要的经济价值的一个植物类群,不仅包括小麦、大麦、黑麦三种重要的粮食作物,而且还有大量具有经济价值的多年生优质牧草,以及对于生态具有重要生态意义的其他物种。由于麦类作物是小麦族物种的一员,它们与小麦族其它物种有着一定的亲缘关系,故小麦族遗传资源作为巨大的基因库,在以遗传操作为手段导入外源有益基因的麦类作物改良中具有重要的潜在价值。事实上,利用小麦族野生近缘属物种中含有的优良基因改良现有小麦品种已经得到了广泛的应用。仲彬草属是一群生长在高海拔地区被毛的小麦族植物,生长于中亚与喜马拉雅地区。一些物种生长在非常严酷的生境之中,可能对禾谷作物的抗寒与抗旱改良作出贡献。偃麦草属是小麦族多年生的野生植物,具有小麦改良的许多重要有益性状,包括免疫或高抗小麦条锈病、叶锈病、秆锈病、白粉病等。因此,仲彬草属和偃麦草属植物与其他多年生小麦族植物一起形成了一个巨大的遗传资源基因库,可以用于一年生禾谷作物的遗传改良。本文围绕小麦族仲彬草属植物的系统进化关系和偃麦草属外源遗传物质的新种质的分子细胞遗传学进行相关研究,为进一步有效合理利用小麦族植物种质资源提供科学指导。其主要的研究结果如下：(1)利用Giemsa-C带技术分析了8个仲彬草属物种,研究结果显示这些物种具有不同的C带核型特征。Kengyilia gobicola、K. alatavica和K. batalinii的染色体具有明显的着丝粒带和不具带纹特征的染色体,而Kengyilia hirsuta、K. longiglumis、K. melanthera、K. rigidula和K. thoroldiana具有丰富的带纹特征,其中包括中间带和端带。不同的带型特征表明了仲彬草属植物在起源和进化上存在分化。(2)利用核基因ITS、alpha淀粉酶抑制因子序列对仲彬草属植物进行系统发育研究,研究结果显示仲彬草属植物与杜威草属、拟鹅观草属、鹅观草属和冰草属植物具有较近的亲缘关系。仲彬草属物种发生了明显的地理分化,地理来源相同或相近的物种亲缘关系较近。不同的鹅观草属物种和冰草属物种可能参与了仲彬草属物种的形成。仲彬草属物种的St和Y基因组具有密切的亲缘关系,但具有不同的起源。(3)对仲彬草属植物的叶绿体atpB-rbcL和线粒体CoxII内含子的核苷酸多态性分析,结果显示仲彬草属植物具有较高程度的核苷酸多态性。叶绿体atpB-rbcL和线粒体CoxⅡ内含子序列的系统发育分析在结果上存在一定的差异,但是总体上仲彬草属物种主要聚为两个分支,分别与不同的二倍体供体物种聚类在一起,表明仲彬草属植物在起源上具有不同的母本供体。(4)对采集于野外疑是属间杂种的小麦族植物进行染色体配对分析和ITS、atpB-rbcL和trnH-psbA序列系统发育分析。结果表明,NHZY6含有St、Y和P染色体组,可能是Kengyilia melanthera与鹅观草属物种的杂交种。NHZY7、NHZY8和NHZY9可能是K. rigidula与Elymus nutans的杂种,而NHZY2、NHZY3和NHZY5是K. melanthera与E. nutans的杂种。基于叶绿体序列的研究结果表明K. rigidula和K. melanthera作为母本供体参与到杂交过程。这些研究结果表明,仲彬草属物种与披碱草属、鹅观草属物种发生了天然杂交,进一步证实了小麦族植物复杂网络化的进化历史。同时,研究支持K. stenachyra是K. rigidula与E. nutans杂交形成的天然杂种的推论。(5)通过对普通小麦与偃麦草的杂交后代进行筛选,对获得的8个渗入系进行分子细胞遗传学研究。结果显示,渗入系染色体数目的变化范围从42到56不等,其中包括44、52、53和54条染色体,单价体频率从0.34到2.36,多价体频率不超过1%,细胞遗传学基本稳定。原位杂交结果表明,小麦的染色体条数从40到44不等,以及数目较多小麦与偃麦草的易位染色体。多色原位杂交的结果显示易位染色体包含了多个基因组之间的相互易位。赤霉病的抗性评价显示,渗入系的感染率从5.65%到11.46%,表明对赤霉病具有高水平的抗性。(6)利用叶绿体trnH-psbA来研究中间偃麦草的遗传多样性和母本起源,结果表明35份中间偃麦草材料形成了11个单倍型,具有较低的核苷酸多态性π0.00473±0.00037和较高的单倍性多态性Hd=0.733±0.061。系统发育分析显示,所有中间偃麦草材料分别与不同的二倍体物种聚类成2个分支,表明中间偃麦草的物种形成过程中至少有两个物种作为母本供体或是该物种经历了多次杂交起源的过程(7)对硬粒小麦与中间偃麦草的新种质08-47-50和08-53-55进行分子细胞遗传学研究。研究结果表明,这两个部分双二倍体含有42条染色体,细胞遗传学上基本稳定,具有不同的染色体组成。08-47-50的染色体组成为28DW+6St+4Ee+4Ee-St,08-53-55的染色体组成为28DW+4St+10Ee-St。这两个部分双二倍体对赤霉病、叶锈病和秆锈病具有高水平的抗性。更多还原

【Abstract】 Triticeae Dumortier is a very important group in the Poaceae which includes three of the most important cereal crops, for example, wheat, barley, and rye, many economically valuable forage grasses as well as a number of significantly ecological importance specices. A clear phylogentic patterns would prompt the ulitization of speices as genetic resources for improving quality and genetic diversity. Because of the affinity between wheat and its relatives, the Triticeae tribe offers a vast gene pool from which traits of agronomic interest can be extracted to enrich the genetic diversity of wheat for disease resistance and quality improvement by genetic manipulation. In fact, a number of relatives to wheat have been to identify to have superior resistance gene and utilized in wheat breeding programme.Kengyilia Yen et J. L. Yang is a recently erected genus grown in high altitude areas. Some species lives in rigorous locality, which can contribute to the improvement of resistance to cold and drought in cereal crops. Elytrigia Desv. is a wild perennial genus in Triticeae tribe. It possesses numerous beneficial traits for wheat improvement including immune or high resistance to tripe rust, leaf rust, stem rust, powder mildew, aphid, salt tolerance and so on. Therefore, Kengyilia and Elytrigia together with other perennial relative species form a huge gene pool for improvement in cereal crops. In this study, the phylogeny of Kengyilia species was evaluated and the molecular cytogenetic characterization of new germplasm materials between wheat and Elytrigia were analyzed on the basis of GISH as well as disease resistance evaluation. The main results are as follows:(1) Giemsa C-banding analysis was implementd to characterize chromosomes of the Kengyilia species. Results indicated that the eight species of Kengyilia differed in C-banding patterns. K. gobicola, K. alatavica and K. batalinii had distinct centromeric bands and no banded chromosomes, while K. hirsuta, K. longiglumis, K. melanthera, K. rigidula and K. thoroldiana had more abundant and diagnostic C-bands with interstitial and terminal bands. The chromosome structural polymorphisims suggested the differentiation regarding the origin and evolution in Kengyilia.(2) Phylogenetic analysis was conducted based on nrDNA ITS and dimeric alpha amylase inhibitor sequences in species of Kengyilia together with those of relatives by the maximum parsimony and neighbor-joining distance methods. The results indicated that species of Kengyilia had close affinities to species of Douglasdeweya, Pseudoroegneria, Roegneria and Agropyron. The species in Kengyilia was identified as two subgroups corresponding to geographic distribution, suggesting that species from the same distribution had a closer phylogenetic relationship. It is possible that the Kenyilia species from Central Asia and adjacent regions and Qinghai-Tibetan Plateau have independent alloploid origin with different P genomic donor. The clear differentiation between the Y and St was detected, which provided additional evidence for the independent origin of the Y genome in alloplooid Roegneria and Kengyilia species.(3) The levels of nucleotide diversity for atpB-rbcL and CoxII intron were obtained. The results revealed relatively high nucleotide diversity. Employing atpB-rbcL and CoxII intron sequences, the phylogenetic relationships within Kengyilia and between Kengyilia and its closely related genera were examined. The maximum parsimony analysis demonstrated that Kengyilia species were positioned into two clades corresponding to different maternal genomic donor. These results indicated that there had been two phylogenetically divergent maternal donors within Kengyilia.(4) Seven accessions of putative natural hybrids between Kengyilia and Roegneria or Elymus from Sichuan and Gansu provinces of China were analyzed using sequences of ITS, atpB-rbcL, trnH-psbA combined with meiosis observation. In summary, NHZY6 was revealed to have St, Y and P genomes, and originated from hybridization between K. melanthera and the species of Roegneria. It is concluded that K. rigidula and E. nutans as parental donors contributed to NHZY7, NHZY8 and NHZY9, and K. melanthera and E. nutans as parental donors contributed to NHZY2, NHZY3 and NHZY5. Chloroplast DNA sequences data demonstrated that K. rigidula and K. melanthera acting as maternal donors were likely to be involved in the formation of the natural hybrids. Also, K. stenachyra was confirmed to be a natural hybrid derived from hybridization between K. rigidula and E. nutans.(5) As a potential source of resistance to Fusarium head blight (FHB), Elytrigia repens has been crossed with common wheat. A total of eight BC1F9 progeny lines were characterized in this study. The chromosome number of these derived lines ranged from 42 to 56, including the lines with 44,52,53, and 54 chromosomes. All of lines were cytologically stable in terms of meiotic configuration. The univalent frequency in these lines varied between 0.34 and 2.36. Similarly, the multivalent frequency did not exceed 1% in any of the lines. GISH analyses revealed that the number of intact wheat chromosomes in the various lines varied between 40 and 44. In all lines, numerous translocated chromosomes were detected, which were involved with chromosomal segments from wheat and El. repens. Trigenomic translocated chromosomes were examined by further investigation. Moreover, the fragments introgressed into wheat chromosomes included both terminal and interstitial type. The FHB resistance of the eight lines, following point inoculation varied from 5.65%infected florets to 11.46%compared to the check cultivars ’Roblin’and’Crocus’at 100%and 85%, respectively.(6) The non-coding intergenic region of trnH-psbA was performed to assess the genetic diversity and infer the maternal origin within Elytrigia intermedia accessions. Eleven haplotypes were distinguished among the thirty-five accessions of El. intermedia. It showed a relatively low nucleotide diversity (π) of 0.00473±0.00037 and a moderately high haplotype diversity (Ha) of 0.733±0.061 within El. intermedia. In the phylogenetic analysis all accessions of El. intermedia were positioned into two clades which were corresponded to the different diploid donors, suggesting that there were two phylogenetically divergent maternal donors in El. intermedia.(7) Two partial amphyploids,08-47-50 and 08-53-55, were developed from wide crosses between durum wheat and El. intermedia. Meiotic analysis indicated that the two lines were cytologically stable, containing 42 chromosomes. Genomic in situ hybridization revealed that the two partial amphiploids carried different chromosomal compositions. Line 08-47-50 had a chromosome complement of 28DW+6St+4Ee+4Ee-St, while the line 08-53-55 had a chromosome complement of 28DW+4St+10Ee-St. The evaluations of Fusarium head blight (FHB), leaf rust and stem rust indicated that the both lines had a high level of resistance.更多还原