【作者】 卢江杰；

【导师】 汤定钦；

【作者基本信息】 浙江林学院 ， 森林培育， 2009， 硕士

【摘要】 毛竹(Phyllostachys pubescens)是禾本科(Gramineae)竹亚科(Bambusoideae)刚竹属(Phyllostachys)植物。是我国竹类资源中最主要的竹种之一,生长快、分布广、利用多、具有重要的经济价值和生态价值。竹亚科植物染色体数目较多而且不稳定,普遍存在多倍性、非整倍性、混倍性和染色体结构变异等现象,染色体结构和基因组特性等方面的研究很少,研究迫切需要一种快速、高效的方法和技术。SSR(Simple Sequence Repeat)标记作为以PCR技术和测序技术为基础的分子标记,是构建高密度分子遗传图谱、物种分类与进化分析、染色体结构分析、品种遗传多样性研究、品种保护与纯度鉴定、研究杂种优势机理及预测的有效工具,被认为是当前种内遗传变异研究中分辨率最高、揭示力最强的分子标记。本研究查阅了大量相关文献,发现竹类植物中的SSR标记很少,只有Nayak和Rout等开发的印度刺竹(Bambusa arundinacea)的6个SSR位点,为了能在竹类植物中开发大量的可以广泛运用的SSR标记,我们选择了重要竹种毛竹为开发对象,尝试在毛竹中开发一批在竹类植物中具有高通用性标记,那样将为其他竹种SSR标记的开发降低成本,为竹类植物相关研究所用。随着网络数据库数据量的日益增加,利用生物信息学的方法来开发SSR引物已渐成熟。本研究通过搜索GenBank毛竹系列,自行开发了19对SSR引物。用这些引物研究了毛竹11个变型(栽培类型)的遗传多样性发现,毛竹和其变型(栽培类型)之间的遗传多样性很低,从电泳图上看所有的19对SSR标记基本上没有什么多态性条带,这与栽培过程中观察到的不同类型之间可以相互转换的现象一致,暗示其变型的产生可能是其他机制造成的。另外,利用刚竹属中7种代表性的竹种对这些SSR引物的通用性进行了分析,发现有10个毛竹SSR标记PBM014, PBM016, PBM018, PBM020, PBM022, PBM025, PBM023, PBM027, PBM028和PBM004在6个刚竹属其他竹种具有很好的通用性;PBM002和PBM007的通用性最低;PBM026虽然有PCR产物,但是不含有SSR位点;其他SSR标记在其他竹种中也有部分不能通用或有扩增产物但是没有SSR位点的现象。总的来说,我们开发的毛竹SSR标记在同属中的通用性为73.7%。由于GenBank中毛竹序列有限,为了得到更多的SSR位点,我们结合传统的构建微卫星富集文库的策略,自行设计了一套新的SSR引物开发策略。使用常用的EcoRⅠ或MseⅠ对毛竹基因组进行酶切,两端分别相对应接上AFLP接头,随后用改进了的少了第一个选择性碱基的AFLP预扩增引物放大酶切片段数量,再用探针(GA)10,(GACA)6,(CA)10,CAA)10,(GAA)10,(CAT)10和(GAT)10杂交和磁珠富集,用改进的引物经PCR后使单链变成双链。片段产物连接pMD18-T载体后转化大肠杆菌,测序。得到的含有SSR位点的序列设计引物,对毛竹基因组DNA进行扩增后,测序预计大小的目的片段,最终我们构建(GA)10和(GACA)6两个探针的文库,初步得到48个毛竹SSR标记。为了进一步研究竹类植物染色体特点,本研究还利用荧光原位杂交(FISH,fluorescence in situ hybridization)技术,以拟南芥端粒重复序列(CCCTAAA)n及寡核苷酸(AG)12、(AT)11、(AAG)5和(GACA)4等作探针,检测这些微卫星序列在毛竹染色体上的分布。掌握了竹类植物染色体上SSR序列的FISH技术,初步了解了拟南芥端粒重复序列(CCCTAAA)n和毛竹染色体上的分布情况,为接下来探索微卫星序列在竹类植物的染色体上的变化,进而推断竹类植物染色体的结构和进化过程开辟了新的研究思路。此外,利用在刚竹属多个竹种中通用性较好的毛竹SSR标记对部分丛生竹以外竹种的杂交后代进行了幼苗期的鉴定,研究杂交竹种中各亲本的遗传物质交流程度。通过实验发现,PBM014,PBM018和PBM025在亲本的SSR位点上等位基因多态性丰富,在亲本和杂交子代上对应位点上的等位基因稳定遗传,表明确实有遗传物质的交流,直接证实了所待测疑似杂种的真实性,为杂交竹种的后期研究工作奠定基础。更多还原

【Abstract】 Phyllostachys pubescens is monopodial bamboo, belonging to the genus phyllostarchys. It is the most important bamboo specie in China, and the most widely distributed with largest planting area (over two-thirds of the total planted bamboo area) and has the highest economic value in the subfamily Bambusoideae within the family Poaceae. There are less researches focus on chromosomal structure and genome character of bamboo, as they reproduced by rhizome in asexual mode and with polyploidy, aneuploidy, mixoploidy and chromosome structure variation. So the studies of bomboo genome need an effective technology, compare with the achievement in other plants. SSR (Simple Sequence Repeat) marker, the new generation marker based on PCR and sequencing, is considered as the powerful tool in high-density molecular genetic mapping, categorization and evolution analysis, chromosome structural analysis, genetic diversity study, germplasm resource protection and purity evaluation, heterosis mechanism study and predication, because of being co-dominant, multiallelic, easily scored and highly polymorphic. There were only 6 bamboo SSR markers in the species of Bambusa arundinacea reported by Nayak and Rout. In order to develop the high transferability bamboo SSR markers for reducing the development cost, we choose Ph. Pubescens first.Only 1, 532 Ph. Pubescens sequences in GenBank. By using bioinformation soft, 19 SSR primer pairs were developed successfully. We used these primers to assess the polymorphism of 1 seeding, 10 cultivars or forms of Ph. Pubescens. At last, we found almost no variation in electromorph size and sequence were detected among the material tested. This survey indicates that these microsatellite loci seem less polymorphism or genetic diversity of Ph. Pubescens is quite lower, and it may explain the phenomenon that different form can change each other frequently. To test the transferability of microsatellites to other bamboo species, six other species of genus Phyllostachys with morphological divergence far away of each others were selected to cross species amplification with SSR primers identified in Ph. Pubescens. Among the 19 SSR loci, 10 loci including PBM014, PBM016, PBM018, PBM020, PBM022, PBM025, PBM023, PBM027, PBM028 and PBM004 completely successfully transferred to six other species of Ph. pubescens. Primers amplifying loci PBM002 and PBM007 in Ph. pubescens barely amplify any PCR products in the other bamboo species. Primer amplifying locus PBM026 amplified PCR product but not containing the SSR motif for the other bamboo species. The other primers partly could not specifically amplify SSR-amplicon or even not amplify any product for the other species. As a result, microsatellite amplification in Ph. pubescens could be across other bamboo species of same genus with 73.7% (84/114) transfer success.As the limited of sequences of Ph. pubescens in GenBank, we designed a new SSR develop strategy based on traditional enriched library construction. Microsatellites were enriched by hybridizing biotin-labeled SSR probes (GA)10,(GACA)6,(CA)10,CAA)10,(GAA)10,(CAT)10 and (GAT)10 with EcoRⅠor MseⅠdigest fragments that amplified by improve pre-amplify AFLP primer (without single elective base primer pairs) after adding AFLP adapters. And captured the hybridized SSR-containing fragments by magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNAs were PCRwith improve pre-amplify AFLP primer. Those became doule-stranded by PCR amplification, ligated into T vector, then transformed into E.coli competent cells to produce a microsatellite enriched library. Sequencing the positive clone which hybridized by blot hybridization. We got more than 48 SSR markers of Ph. pubescens by probes (GA)10 and (GACA)6 at last.Aim to study the chromosomal structure of bamboo, we detected the the Arabidopsis-type telomere sequence (CCCTAAA)5 and oligo (AG)12, (AT)11, (AAG)5, and (GACA)4 probes. Though didn’t got the perfect pictures of SSR physical map, it is the first time we use FISH (fluorescence in situ hybridization), and study the distribution of (CCCTAAA)5 and oligo (CA)10, (GA)10 premier, which break the new way in bamboo SSR physical map and chromosomal structure.The different number copy in SSR motifs bring about the SSLP (single sequence length polymorphism) among bamboo species and the loci may be useful for genetic analysis and identification of hybridization bamboo. The SSR marker PBM014,PBM018 and PBM025 have abundance allele polymorphism, and inherited stabile in hybridization offspring by sequencing the SSR loci, all the results show that the hybrids of bamboo in this study are true. Establish the foundation for further research.更多还原