【作者】 刘贯水；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2010， 博士

【摘要】 竹类植物是禾本科一个最大的自然类群,具有70余属,1200余种。竹子不仅具有生长快,用途广等特性,而且还具有良好的水源涵养和水土保持等环境功能。竹类植物通常难以开花,而且一旦开花,植株乃至全林即全部枯死,因而传统的以花、果实形态为主的分类方法对竹类植物十分困难。目前竹子的分类依然存在着很多争议,而分子标记技术的应用为竹子系统分类的深入开展提供了新的依据。SSR(Simple Sequence Repeat)简单序列重复,又称为微卫星,这类分子标记具有含量丰富,多态程度高,检测方法简便,不受环境和基因表达影响等特点,在种质鉴定、遗传多样性分析以及系谱分析中成为一种理想的分子标记。目前,竹类植物的微卫星引物开发国内外报道较少,无法满足竹子系统研究的需要。本研究通过磁珠富集法开发了31对毛竹SSR引物并对部分竹类植物进行了系统分类学研究。主要结果如下:1通过磁珠富集,成功构建了毛竹基因组DNA的GT、AG、CCA重复序列的富集文库,利用克隆PCR方法共检测了2330个克隆,三个文库分别检测菌落数为1080、620、630个,阳性克隆分别为219、113、119个,阳性率为19.4% (451/2330);2获得350条非冗余序列,长度范围在61~831bp之间,平均为288bp,序列总长为100 704bp,占毛竹基因组大小约1/20000;发现重复位点641个,重复次数在3~54次之间,其中重复3、4、5、6、7、8、9、10、>10次的位点数分别为415、86、37、14、19、8、12、11、39个;双核苷酸重复位点最多,为521个,三核苷酸、四核苷酸位点数分别为108、35个;3对获得序列进行Blast比对,结果显示:所得序列与GenBank登录序列的毛竹基因序列相似数最多,为63条,与水稻、甜高粱、小麦、玉米序列相似数分别为28、11、3、3条,这与毛竹的基因组进化地位相吻合;4设计合成了53对SSR引物,以毛竹基因组DNA为模板对这些引物是否工作进行了检测,其中34对引物扩增出了预期大小的条带,利用温度梯度PCR确定了各引物最适退火温度;5用荧光标记物FAM对34对引物中的正向引物进行标记,利用毛细管电泳分析9份毛竹种内样品SSR-PCR扩增产物的大小,其中引物对27、36、40的扩增产物中具有非预期大小的片段,对这三对引物扩增黄槽毛竹的PCR产物进行了克隆和序列分析,发现这些位点存在序列重复;利用其余31个位点对9份毛竹种内样品进行位点特征分析,发现31个位点在除黄槽毛竹的8个毛竹样品中都有预期大小的等位基因的扩增,而黄槽毛竹的位点特征与其他8个毛竹样品具有较大差异,其中位点2、17、38、42、50对黄槽毛竹显示零等位,位点8、13、15、18、21、24、28、31、32、33、37、41、43、46、49、51具有不同于其他8个毛竹样品的特殊等位基因,这些差异位点占总位点数的61.8% (5+16/34);6对除黄槽毛竹的8份毛竹样品的31个SSR位点进行分析,结果显示:有18个位点在所有8份毛竹样品中都只扩增出了一个大小相同的片段,其余13个位点的扩增条带具有差异,表现出了多态性;这31个位点的等位基因数(Observed Number of Alleles, Na)在1~3个之间,平均为1.548个;有效等位基因数(Effective Number of Alleles, Ne)在1~2.65之间,平均有效等位基因数为1.375个;这些位点的观测杂合度(Observed Heterozygosities, Ho)在0~1之间,平均为0.309;期望杂合度(Expeeted Heterozygosities,He)在0~0.67之间,平均为0.201;多态信息含量在0~0.575之间,平均为0.147;13个多态位点中只有1个位点的多态信息含量在0.5以上,其余12个位点在0.110~0.48之间,平均为0.352,多态信息含量较低,这些都表明毛竹种内遗传差异较小,基因突变程度较低;7对唐竹属、业平竹属、大节竹属的11个竹种的31个SSR位点进行了分析,检测结果用NTsys软件计算竹种间的Nei氏遗传距离(Nei’s Distance),非加权算数平均法(Unweighted Pair-group Method with Arithmetic Means)对遗传距离矩阵进行聚类分析,结果显示31个SSR位点可以将这11个竹种分为三个属区;8对刚竹属内51份样品的31个SSR位点进行了分析,聚类结果表明:符合传统分类学方法,说明这些SSR位点可以为刚竹属植物属内的分类提供参考;各竹种种下等级之间的遗传距离大于零,说明它们相互间存在着一定的遗传变异,个体间具有遗传多样性,表明这些SSR位点可以对种内个体的进行遗传多样性及聚类研究,而且利用这些引物也可以开发为竹种或杂交种分子鉴定的标记;结果还显示竹种的种内变异程度小于种间变异,表明这些SSR位点可以为刚竹属的存在争议的种的界定提供参考。更多还原

【Abstract】 Bamboo (Bambusoideae), the largest subfamily of the grasses (Poaceae), contains about 70 genera and 1200 species. Bamboo, the fastest growing and most widely-used woody species in the world, which is of economic significance, furthermore it plays an important role in the function of water conservation and soil conservation. It is difficult to classify and identify bamboos by using the traditional taxonomy method, which is based on the forms of flowers and fruit, because they seldom flowering and once it happens the plant or even the whole bamboo forest become dead. At present, the current bamboo classification is always in dispute in the academic field, and the application of molecular marker technology provide the basis for bamboo taxonomy. Simple Sequence Repeat or Microsatellite marker is an ideal tool for germplasm identification, genetic diversity and pedigree analysis, because they are abundant in genomic DNA, high polymorphism, easy to detect and uninfluenced by environment and gene expression. However, seldom bamboo microsatellite marker have been reported.This research developed 31 pairs of bamboo SSR primers by using the method of microsatellite enrichment of magnetic beads and analyzed 62 samples in 4 genera. The results are as follows:1. Three microsatelite enriched libraries (GT, AG, CCA) were constructed from Ph. heterocycla var. pubescens (Mazel) Ohwi genomic DNA, 2330 clones were screened by colony-PCR, with 1080, 620, 630 clones respectively, 219, 113, 119 positive clones were sequenced, the ratiowas 19.4% (451/2330).2. 350 non-redundancy sequences were obtained, which were between 61 bp and 831bp. The average length was 288bp, and the total length was 100 704bp, about 1/20 000 the size of Ph. heterocycla var. pubescens (Mazel) Ohwi genomic DNA. 641 SSR sites were found, the number of repeat motifs in each site were between 3~54, with 415, 86, 37, 14, 19, 8, 12, 11, 39 sites of 3, 4, 5, 6, 7, 8, 9, 10, >10 repeat unit. The sites of dinucleotide composed the most, with 521 sites, trinucleotide, tetronucleotide were 108, 35 sites respectively. 3. Blast analysis of obtained sequences showed that: 63 sequences producing significant alignments to Ph. edulis sequences in GenBank, composed the most, and 28, 11, 3, 3 sequences producing significant alignments to rice, sweet sorghum, wheat and corn respectively, which consistent with the phylogenetic position of Ph. heterocycla var. pubescens (Mazel) Ohwi.4. 53 pairs of SSR primers were designed, 34 of which amplified target fragment from Ph.heterocycla var. pubescens (Mazel ) Ohwi genomic DNA and the annealing temperature were determined by using temperature gradient PCR.5. FAM labeled forward primer of 34 pairs of primers, capillary electrophoresis analyzed the length of SSR-PCR product of 9 samples of intraspecies of moso bamboo, loci 27, 36, 40 amplified specific fragments, clone and sequenced several fragments of these three primers’ PCR amplification product in Ph. heterocycla cv. Luteosulcata. The result showed that, these loci manifested gene duplication. The other 31 loci analysis of 9 species showed that 31 loci amplified expected alleles at all 8 samples of moso bamboo except Ph. heterocycla cv. Luteosulcata. Ph. heterocycla cv. Luteosulcata manifested difference to the other 8 samples. Loci 2, 17, 38, 42, 50 showed null allele, loci 8, 13, 15, 18, 21, 24, 28, 31, 32, 33, 37, 41, 43, 46, 49, 51 ampified specific alleles to other 8 species, these loci takes the rate at 61.8% (5+16/34).6. 31 paris of developed SSR markers were used to assess the genetic diversity among the 8 moso bamboo, 18 loci were amplified only one band, 13 loci were polymorphism, at the rate of 41.94 %, the observed number of alleles were between 1~3, at an average of 1.548, and the effective number of alleles were between 1~2.6, at an average of 1.375; the Observed heterozygosities and Expeeted heterozygosities were between 0~1, 0~0.67, at an average of 0.309, 0.201. The PIC (Polymophism Information Content) is between 0~0.575, at an average of 0.147, showed the low level of mutation in these 8 moso bamboo.7. Capillary electrophoresis analyzed 31 SSR loci in 11 species belongs to 3 genera Semiarundinaria,Sinobambusa,Indosasa, Ntsys software calculated Nei’s genetic distance and clustering analyzed by unweighted pair-group method with arithmetic means, the result showed that these 31 loci could distinguish these species 3 genera. 8. Capillary electrophoresis analyzed 31 SSR loci in 51 species (varieties or types) of genus Phyllostachys, the result showed that: On the whole, the clustering result consistent with the traditional taxonomy, shows that these SSR loci could be used to classify the genetic distance of Phyllostachys; the genetic distance between intraspecies is larger than 0, shows the presence of genetic variation and genetic polymorphism between them, so these loci could developed for species identification markers; the result also shows the genetic distance between species is larger than intraspecies, so these loci also could used for defining species which is in disputing.更多还原