【作者】 蔡丹英；

【导师】 滕元文； David M.Spooner；

【作者基本信息】 浙江大学 ， 果树学， 2011， 博士

【摘要】 马铃薯(Solanum tuberosum L.)属于Solanum属中的section Petota Dumortier。其中Petota组中36%为多倍体。已有学者采用质体DNA限制性位点、颗粒结合型淀粉合成酶(GBSSI)、硝酸还原酶(NIA)、直系同源COSⅡ标记等方法对马铃薯的二倍体和多倍体进行了系统发育关系研究,但这些研究由于只采用了较少的基因或标记抑或是较少的多倍体物种,而并没有对多个物种的野生多倍体马铃薯进行全面的系统发育分析。本研究采用6对COSⅡ标记对11个野生多倍体物种中的54个个体以及33个野生二倍体物种中的37个个体进行了PCR扩增,利用优化的非对称PCR-SSCP技术对所得到的等位基因进行测序并进行较全面的野生多倍体马铃薯的系统发育研究,表明野生多倍体马铃薯中可能存在多起源现象；同时和采用其它基因得到的结果相比较,结果相似。主要研究结果如下：1.获得了最优化的非对称PCR-SSCP方法的条件,确定了不同的COSⅡ标记各自最佳的非对称PCR-SSCP条件。和传统的克隆测序方法比较,非对称PCR-SSCP方法省时,省力,可以避免克隆测序中易发生的PCR重组以及异源双链核酸分子,并且其多态性位点的碱基类型和数目均和克隆测序结果一致,在某种程度上可以替代克隆测序。对于多倍体而言,成本可以降低到原来的18.9%-33.3%。。2.将优化的非对称PCR-SSCP方法推广到6对COSⅡ标记以及11个物种的54个野生多倍体和33个物种的37个野生二倍体马铃薯个体中,主要用于研究野生多倍体马铃薯的系统发育关系。单个COSH的比对长度介于461(C2_Atlg32130)～1473 (C2_Atlg20050)个位点之间,简约信息位点数介于45-134个之间,一致性指数(Consistency Index, CI)介于0.73-0.86之间,保留指数(Retention Index, RI)介于0.94-0.97之间。5个COSⅡ(不包含C2_At5g47390序列数据)和6个COSⅡ联合序列数据中,序列比对总长度分别为4031和4719个位点,简约信息位点数分别为673和551个,一致性指数(CI)和保留指数(RI)均为0.63和0.92。对二倍体序列的分析结果表明,在222个组合(37个二倍体×6个COSⅡ)中,12.6%含有多于1个的等位基因。排除差异较小的等位基因后,只有5.9%的二倍体含有差异。四倍体用于最终分析时,只有1.8%的个体少于两个等位基因；六倍体亦是如此,只有1.8%的个体少于三个等位基因。3.采用最大似然法对不同的序列数据进行系统发育分析。用最大似然法对单个COSⅡ标记的二倍体序列分析表明,虽然这6个单独的COSⅡ得到的系统树存在着不一致,但可以很好地解决二倍体物种的系统发育关系。用最大似然法对二倍体和多倍体的6个COSⅡ联合序列数据分析得到和前人不一致的研究结果,它将分枝3分成两小分枝。但对含有5个COS联合序列文件进行最大似然法分析时,得到和前人相似的研究结果,包含三个主要的分枝分枝1+2,分枝3,分枝4。4.用最大似然法对二倍体和多倍体的5个COSⅡ联合序列数据分析表明,54个多倍体中有29个多倍体的等位基因在系统树中所在分枝和GBSSI、NIA所得到的系统发育结果相符：所研究的多倍体均为为异源多倍体起源；多倍体和亲缘关系较近的二倍体在同一分枝上。25个多倍体个体中显示出了不同于前人的研究结果：1)多倍体基因在起源不明的分枝3p、4apl和4p2中,该分枝没有包含二倍体物种的等位基因；2)在原有的三大分枝上发现新的多倍体基因；3)有23个物种/COS组合等位基因发生明显的片段丢失,存在某些系统发育上的相关性；4)野生多倍体马铃薯中存在多起源现象。更多还原

【Abstract】 Solanum tuberosum L.belongs to Solanum L. section Petota Dumortier, of which 36% in sect. Petota were polyploid. Many different kind of enzymes or markers were used for the phylogeny of wild diploid and polyploid potatoes, e.g., plastid DNA restriction site, Granule-bound starch synthase (GBSSI), Nitrate Reductase (NIA), Conserved Ortholog setⅡ(COSII). But not so much wild polyploid potatoes were used in these researches. We here conduct a DNA sequencing study, using six nuclear orthologs, of 54 accessions of 11 polyploid species, and 37 diploid species representing possible progenitors to address questions species and genome origins of the polyploids, possible multiple origins, and concordance of different molecular markers from different regions of the genome. The main results were as follows:The protocols of asymmetric PCR-SSCP were optimized by different kind of tests, later the optimized protocols for each individual COSII were set up. Compared to the traditional cloning, the optimal asymmetric PCR-SSCP protocols saved time and labors, decreased PCR recombination and heteroduplex molecules during PCR processing and had the same base types and base numbers in polymorphic sites with clone sequencing results. In some extent, it could replace cloning method. Also the cost could be reduced by 18.9%-33.3% for polyploid species.The optimal protocols and six COSII were applied in all the materials in this study to construct phylogeny of the wild polyploid potatoes. The aligned length of the individual six COSII ranged from 461 characters for C2_Atlg32130 to 1473 for C2_At1g20050. The concatenated length of all six COSII was 4719 characters and for five COSII 4031 (without C2_At5g47390), of which parsimony informative characters were 673 and 551 respectively. The consistency indices of the individual COSII datasets ranged from 0.73-0.86 and the retention indices from 0.94-0.97. The consistency/retention indices were lower for both concatenated dataset,0.63/0.92. Of the 222 combinations (37 diploid accessions x 6 COSII),12.6% had more than one minor variant allele. After elimination alleles with minor allelic variants, only 5.9% of the diploids had allelic variants. Only 1.8% of the alleles from the tetraploids had less than two retained (divergent) alleles and only 1.8% the hexaploids had less than three divergent alleles.The phylogenetic analysis were performed by using different kind of datasets (six individual COS datasets of diploid+polyploid, six-COSII concatenated dataset of diploid, six-GOSII concatenated dataset of diploid+polyploid, five-COSII concatenated dataset of diploid and five-COSII concatenated dataset of diploid+polyploid). The results of six individual COS datasets of all diploid and polyploid indicated Though various degrees of incongruence were found in all six individual COSII phylogenetic trees, the diploid phylogeny was well solved. The 6COS concatenated dataset of diploid+polyploid result differs from prior phylogenies, in separating species from clade 3 into two clades. However, removing data from COSII C2At5g47390 from the dataset (retaining data from the remaining five COSII) recovered a three clade topology as did all three prior results.The five-COSII concatenated dataset of diploid+polyploid results concurred with the cladistic relationships of potato allopolyploids using GBSSI and NIA in 29 of the 54 cases examined:all polyploids were allopoplyploid origins; polyploids and more related diploids were in the same clades The remaining 25 of 54 accessions show three classes of discordance not seen in prior results:1) polyploid alleles in clades of unknown origins not recovered before (3p,4ap1,4p2), without diploid alleles in these new clades; 2) new polyploid alleles in the prexisting three main clades, relative to the studies of GBSSI and NIA; 3) 23 accession/COS combinations, alleles were apparently lost and phylogenetic associations stand out, and 4) multiple origins were found in wild polyploid potatoes.更多还原