【作者】 张鹏；

【导师】 张翠翠； 范优荣；

【作者基本信息】 广西大学 ， 生物学， 2021， 硕士

【摘要】 分子标记辅助选择(Molecular Marker-Assisted Selection,MAS)是育种家们在研究过程中提升育种筛选效率、增加目标基因筛选准确性、方便快捷得到优良品种的重要策略。随着分子检测技术的飞速发展,在大数据的时代背景下,高通量的单核苷酸多态性(SNP)标记凭借其数量多、通量高、规模大的特点,在育种过程中得到了广泛应用。本实验室前期通过改良转座子显示技术,发明了一种新的高通量SNP分子标记检测方法。该方法利用一条转座子引物和一个前景基因的特异引物,通过一步Tn5转座酶反应和两轮PCR扩增构建一个二代测序文库,分析其测序数据可以得知检测样品的前景基因型和遗传背景。在此基础上,本研究对其实验流程进行改良优化,利用特异引物在基因组模板上完全匹配的退火(primer-template perfect annealing,PTPA)扩增目标片段作为前景标记,同时利用引物在基因组模板上的不完全匹配的退火(primer-template mismatches annealing,PTMA)扩增出数以十万计的其它片段作为全基因组的背景标记,最终开发出可以一次性同时检测多个目标前景位点和全部遗传背景的FBI(Foreground and Background Integrated genotyping)技术,该技术不受物种限制,可为育种家的种质研究工作提供更多便利。具体来说,本研究共获得了以下结果:1.完成一个前景基因到多个前景基因筛选的实验方法优化,使引物的PTMA和PTPA扩增效应更强,通过Q-PCR检测最终确立了FBI方法的最优体系。2.完成了FBI技术不同引物在不同作物品种的通用性研究,证明了该方法不受物种的限制,任何一条引物都可以完成不同作物的遗传背景标记。3.利用高保真酶和非高保真酶对比实验,确定了FBI技术特异引物的PTMA效应的匹配模式是引物中间的5～7碱基完全匹配,并且是以滑窗式移动的结合扩增的方式。4.在FBI-seq的应用研究中,完成了同时对稻瘟病抗性基因Pi2、香味基因BADH2、育性恢复基因Rf3和Rf4、品质基因Waxy的5个前景的46个高世代株系的遗传前景和背景的育种筛选。这些实验结果表明,FBI-seq仅需要一条不需要特殊设计和优化的普通引物即可实现对前景标记和数十万个位点的背景标记的同时检测,且能够很方便的转换到不同的前景基因和不同的物种中,尤其是基因组信息积累较少的物种上。此外,该方法可以同时进行多达6种前景的分子标记检测,相比目前其它全基因组标记检测方法,本研究提出的基于LM-PCR和NGS测序相结合的FBI技术,实验流程简单,是一种简单快捷且符合潮流趋势的新型育种的全基因组分子标记方法,在育种研究的基因型检测中具有广泛的应用前景。更多还原

【Abstract】 Molecular Marker-Assisted Selection(MAS)is an important strategy for breeders to improve the efficiency of breeding screening,increase the accuracy of target gene screening,and obtain high-quality varieties conveniently and quickly during the research process.With the rapid development of molecular detection technology,in the era of “big data”,high-throughput single nucleotide polymorphism(SNP)markers have been widely used in breeding research due to their characteristics of great quantity,high-throughput,and a large scale.Our laboratory earlier invented a new high-throughput SNP molecular marker method through improving transposon display technology.The method was using a transposon primer and a specific primer for the foreground gene to generate a second-generation sequencing library through one-step transposase reaction and two rounds of PCR amplification,and the sequencing data can be analyzed to know the target genotype and genetic background of the tested sample.Based on this,the present study improved and optimized the experimental procedure,using primer-template perfect annealing(PTPA)to amplify the target fragment as the foreground markers while using the primer-template mismatches annealing(PTMA)to amplify hundreds of thousands of other fragments as background markers for the whole genome,and finally developed FBI(Foreground and Background Integrated genotyping)technology that can simultaneously detect multiple target foreground loci and the whole genetic background.This technology is not restricted by species,and which can provide more convenience for the research work of breeders.Generally,the following results were obtained in research:1.Complete the optimization of the experimental method for screening one foreground gene or multiple foreground genes,so that the PTMA and PTPA amplification effects of the primers are stronger,and the final optimal system of the FBI method is established through Q-PCR detection.2.The versatility research of different primers of FBI technology in different crop varieties has been done,which proved that the method is not restricted by species,and any one primer can get the genetic background marking for different crops.3.Using high-fidelity enzyme and non-high-fidelity enzyme comparison experiments,it is determined that the matching mode of the PTMA for the specific primer in the FBI technology,and the mode is the5-7 bases in the middle of the primer matching perfectly with a sliding window moving combined amplification method.4.In the application research of FBI-seq,we have tested the whole background and 5 foreground genes of blast resistance gene Pi2,aroma gene BADH2,fertility-restoration genes Rf3 and Rf4,and quality gene Waxy in 46 rice lines.These results show that the method of FBI-seq only needs a common primer that does not require special design and optimization to realize the simultaneous detection of the foreground marker and hundreds of thousands of background markers,and can easily switch to different foreground genes and different species,especially those with less genomic information.In addition,this method can simultaneously detect up to 6target foreground genes.Compared with other current whole-genome marker detection methods,the FBI technology based on the combination of LM-PCR and NGS sequencing proposed in this study has simple experimental procedures and is a kind of simple,fast and trendy new-type breeding genome-wide molecular marker method,and has wide application prospects in the genotyping for breeding research.更多还原