【作者】 王真慧；

【导师】 刘宝；

【作者基本信息】 东北师范大学 ， 遗传学， 2013， 博士

【摘要】 种间杂交(inter-specific hybridization)在植物进化过程中发挥了重要作用。杂交后代中所产生的基因组冲击（genomic shock）能诱导其在遗传水平产生突变，而新产生的遗传变异将会直接或间接地导致杂交后代产生新的表型，以最终通过增加杂交后代的适应度（fitness）以更好地适应变化的环境。为了从全基因水平探讨种间杂交诱导遗传变异的规律，并在此基础上揭示新产生的变异导致杂交后代表型改变的分子机制，本研究采用基因组重测序技术对栽培水稻松前（Oryza sativa ssp. japonica cv. Matsumae）及其菰渐渗系RZ35进行了高通量测序。数据分析显示，本实验中共产生了1.31亿的reads，共计117.9亿个碱基对。这些reads分别覆盖了13.2倍松前的基因组和21.9倍RZ35的基因组。在进一步的分析中，通过比对渐渗系RZ35和其母本松前的基因组，我们鉴定出了41724个纯合单核苷酸多态性（single nucleotide polymorphisms, SNPs）和17839个纯合小片段插入缺失位点（insertion/deletion, INDEL）。通过对SNPs在基因组中的分布情况进行统计显示，这些SNPs趋向于集中分布于染色体上的不同区域，从而形成了一些高突变区（high mutation regions, HMRs）。此外，通过与水稻驯化相关区域进行对比分析发现，其中有11个HMRs与驯化相关区域重叠，该结果显示这些区域可能更容易受到种间杂交的诱导。基于以上结果，我们对所获得的SNPs进行功能分析发现有3797个SNPs为非同义（nonsynonymous）突变，这些SNPs广泛分布于2250个基因中。为了近一步验证这些突变基因对RZ35表型改变的贡献，我们将这些基因定位了到各个代谢途径（pathway）中。分析结果表明，部分突变基因集中分布在几个与植物抗逆相关的途径中，例如糖酵解途径、脂肪酸合成途径、苯丙氨酸合成途径等。该结果证明种间杂交所诱导的突变可以诱导杂交后代产生新的表型变异，而这些表型变异增加了植物本身对逆境的抗性。此外，我们还对RZ35全基因组范围内转座子激活进行了分析。结果表明，相对于SNPs在RZ35染色体上集中分布的而言，本实验中所鉴定的11个转座子家族在不同染色体上随机产生了63次转座激活，例如mPing、Osr29、Tos17等。为了进一步验证转座子激活对RZ35基因功能的影响，我们对被激活的转座子所在的具体区域进行了分析，发现部分转座子插入到基因结构区及其上游1kb范围内。例如，一个mPing转座子插入到LOC_Os05g11130基因的内含子中。该结果表明种间杂交所诱导的转座子激活可能会导致部分基因的功能发生改变，并可能导致杂交后代的表型也随之发生改变。为了验证新产生的表型是否对杂交后代适应环境有贡献，我们对松前和RZ35进行了稻瘟病菌（Magnaporthe grisea）接种实验。该实验结果显示，RZ35具有相对高的抗稻瘟病表型。通过对多个抗稻瘟病主效基因的表达量进行qRT-PCR检测发现，在RZ35中的Pid3/Pi25的表达水平明显上调。此外，我们发现在该基因的编码区发生了一个非同义突变（由丝氨酸突变成酪氨酸）。因此，我们推测该突变可能是导致RZ35具有高抗稻瘟病特性的主要原因之一。综上所述，本文研究结果证明有性不亲和植物种间杂交（通过发生染色质小片段渐渗）诱导RZ35产生了大量的遗传变异，而部分遗传变异导致了一些新表型的产生，并以此提高杂交后代的适应度。因此，本研究不仅证实了有性不亲和植物种间杂交对植物进化可能存在较大的贡献，同时也表明了其也是一种潜在和高效的作物遗传改良方法。更多还原

【Abstract】 Hybridization between genetically diverged organisms is an important avenuethat drives plant genome evolution. The possible outcomes of hybridization would bethe occurring of genomic shock‘‘which could evoke the genetic variation in thederived hybrids. The new induced variations may result in phenotypic noveltiesdirectly or indirectly, and eventually increase the fitness of the resulted hybrids.To address the molecular mechanism of how the inter-specific hybridizationcauses the genetic variation and to what extent it leads to the phenotypical novelties atwhole genome level, we re-sequenced the genomes of Oryza sativa ssp. japonica cv.Matsumae and one of its derived introgressant RZ35that was obtained from anintrogressive hybridization between Matsumae and Zizania latifolia Griseb.According to the results, our study generated a total of131millions90base pair (bp)paired-end reads which covered13.2and21.9folds of the Matsumae and RZ35genomes, respectively. In addition, a total of41,724homozygous single nucleotidepolymorphisms (SNPs) and17,839homozygous insertions/deletions (INDELs) wereidentified in RZ35. Notably, we found that these SNPs tended to occur in thechromosome as clusters and thus a number of high mutation regions (HMRs) wereidentified in our study. Interestingly, we found that11HMRs were located within thedomesticated related regions, implying that the HMRs may fragile and hence labile tomutations as a result of introgressive hybridization.In addition, our results revealed that3,797SNPs were nonsynonymous mutationswhich randomly distributed in2,250genes. To further investigate how these mutatedgenes contributed to phenotypic novelties of RZ35, we submitted these genes intoKEGG and identified that some of these mutated genes were concentrated in severalphysiologically important pathways including glycolysis/gluconeogenesis, fatty acidbiosynthesis and phenylpropanoid biosynthesis pathways. The results yield theconclusion that inter-specific hybridization has indeed resulted in the phenotypicnovelties that enhance the abilities of plant to tolerance the stresses.In addition to the observed genetic variation, rampant mobilization oftransposable elements (TEs) was also identified in the RZ35genome. Notably, a totalof63mobilization events representing11TE families were found including mPing,Osr29, Tos17and so on. To further examine how the mobilization of TEs affects thefunctions of genes, we analyzed the locations of each TE insertions. For example, we found that a member of mPing family was inserted in the intron regions ofLOC_Os05g11130. It indicates that inter-specific hybridization can cause theactivation of TEs and some of which may result the alterations in gene function andeventually lead to phenotypic novelties of resulted hybrids.To examine how the phenotypic novelties contributed to the fitness of hybrids,we performed the inoculation experiment between Matsumae and RZ35usingMagnaporthe grisea. Results from pathogen inoculation revealed that RZ35exhibitedenhanced resistance to blast relative to cv. Matsumae. Notably, one nonsynonymousmutation was identified in Pid3/Pi25. This finding coupled with the results ofreal-time quantitative (q) RT-PCR illustrated that it may be responsible for theenhanced resistance to rice blast in RZ35.Our results demonstrated that introgressive hybridization by alien pollens of evena sexually incompatible species may represent a potent means to generate novelgenetic diversities, and which may have played relevant roles in plant evolution andcan be manipulated for crop improvements.更多还原