石刁柏雄性偏向核质体DNA的克隆与分析

该研究以雌雄异株植物石刁柏为材料,利用基因组消减杂交技术对石刁柏雌雄核基因组中的性别差异核质体DNA（nuclear plastid DNA,NUPTs）进行了分离和分析。结果表明：（1）通过构建消减杂交文库共获得了52个雄性偏向序列,序列长度分布在63~297 bp之间,其中有19个差异序列属于叶绿体来源序列（命名为Ao1~Ao19）,且这些序列与石刁柏叶绿体基因组的相似性均大于84%,Ao19与石刁柏叶绿体基因组相似性为100%。（2）利用基因组半定量PCR对19个NUPTs序列的性别差异分析表明,有4条序列为稳定的雄性偏向NUPTs序列,分别为Ao1、Ao3、Ao10和Ao18。（3）序列比对表明,转移到核基因组的NUPTs主要来源于叶绿体基因组的反向重复区（包含IRa和IRb区）,说明石刁柏叶绿体基因组重复区序列更容易向核基因组进行转移形成雄性偏向的NUPTs序列。     

Assembly-free quantification of vagrant DNA inserts

Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called “vagrantDNA” and code to simulate test data sets.     

Patterns of Genomic Integration of Nuclear Chloroplast DNA Fragments in Plant Species

The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts.     

石刁柏核质体DNA的生物信息学分析及染色体定位

在植物基因组中, 叶绿体DNA (cpDNA)序列可以向核基因组转移成为核质体DNA (NUPT)。NUPTs在植物染色体(包括性染色体)的演化过程中具有重要作用, 但目前相关研究比较缺乏。以雌雄异株植物石刁柏(Asparagus officinalis)为材料, 采用生物信息学方法对其核基因组NUPTs进行注释及分析, 并选取叶绿体基因组反向重复区(IR) 2个片段进行染色体定位。结果表明, 石刁柏核基因组中有2 239个NUPTs序列的插入, 总长度为565 970 bp, 占核基因组的0.047%。不同染色体上插入的NUPTs数量存在较大差异, Y染色体上的NUPTs数量、密度及总长度均高于其它染色体, 表明NUPTs在石刁柏性(Y)染色体上累积的更多。石刁柏叶绿体基因组中的IR区、大单拷贝区(LSC)和小单拷贝区(SSC)序列均能够向核基因组转移, 但IR区序列转移频率更高。此外, 对2个IR区的叶绿体序列进行荧光原位杂交, 其中AocpIR1主要分布在所有染色体的着丝粒部位, 而AocpIR2特异性分布在Y染色体上。研究结果为深入揭示石刁柏基因组的结构及其性染色体的演化奠定了坚实的基础。     

石刁柏核质体DNA的生物信息学分析及染色体定位

在植物基因组中, 叶绿体DNA (cpDNA)序列可以向核基因组转移成为核质体DNA (NUPT)。NUPTs在植物染色体(包括性染色体)的演化过程中具有重要作用, 但目前相关研究比较缺乏。以雌雄异株植物石刁柏(Asparagus officinalis)为材料, 采用生物信息学方法对其核基因组NUPTs进行注释及分析, 并选取叶绿体基因组反向重复区(IR) 2个片段进行染色体定位。结果表明, 石刁柏核基因组中有2 239个NUPTs序列的插入, 总长度为565 970 bp, 占核基因组的0.047%。不同染色体上插入的NUPTs数量存在较大差异, Y染色体上的NUPTs数量、密度及总长度均高于其它染色体, 表明NUPTs在石刁柏性(Y)染色体上累积的更多。石刁柏叶绿体基因组中的IR区、大单拷贝区(LSC)和小单拷贝区(SSC)序列均能够向核基因组转移, 但IR区序列转移频率更高。此外, 对2个IR区的叶绿体序列进行荧光原位杂交, 其中AocpIR1主要分布在所有染色体的着丝粒部位, 而AocpIR2特异性分布在Y染色体上。研究结果为深入揭示石刁柏基因组的结构及其性染色体的演化奠定了坚实的基础。