Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping

TILLING (Targeting Induced Local Lesions IN Genomes) exploits the fact that CEL I endonuclease cleaves heteroduplexes at positions of single nucleotide or small indel mismatches. To detect single nucleotide polymorphisms (SNPs) across a population, DNA pools are created and a target locus under query is PCR-amplified and subjected to heteroduplex formation, followed by CEL I cleavage. Currently, the common method used to detect cleaved products is by polyacrylamide gel electrophoresis using a high-throughput genotyping platform. Exact SNPs are then determined by sequencing. We sought to simplify the detection of CEL I-cleaved products on conventional agarose gels to make the technique more accessible to collaborating partners in developing countries where access to instrumentation could be limiting. Here, we used a panel of stress-related genes to evaluate SNP detection across 48 rice genotypes by contrasting them individually against IR64 and Nipponbare. SNP detection calls corresponded perfectly with those obtained from the Li-Cor genotypers. We were able to detect SNPs in pools of eight DNA templates, suggesting that the agarose gel system could be used to screen for SNPs with comparable throughput as that of the Li-Cor genotypers and showed that the throughput can be increased by analyzing larger amplicons (∼3 kb). The agarose method offers a significant advantage by alleviating the need for labeled primers. We further demonstrated that the agarose method can be effectively used in gene mapping, an application particularly useful for parental lines with low levels of polymorphism. The lower cost and simplicity of the technique make it possible for broader applications of SNP-based markers for germplasm characterization and mapping studies.     

TILLING技术及其应用

定向诱导基因组局部突变(targetinginducedlocallesionsingenomes,TILLING)可快速、有效地鉴定和定向筛选突变,是一种全新的反向遗传学技术。现对TILLING的技术流程、核心与特点,及其在突变研究、反向遗传学及功能基因组学、SNP检测、资源创新与分析以及作物遗传改良等方面的应用进行了综述。     

TILLING moves beyond functional genomics into crop improvement

Transgenic methods have been successfully applied to trait improvement in a number of crops. However, reverse genetics studies by transgenic means are not practical in many commercially important crops, hampering investigations into gene function and the development of novel and improved cultivars. A nontransgenic method for reverse genetics called Targeting Induced Local Lesions IN Genomes (TILLING) has been developed as a method for inducing and identifying novel genetic variation, and has been demonstrated in the model plant, Arabidopsis thaliana. Recently, TILLING has been extended to the improvement of crop plants and shows great promise as a general method for both functional genomics and modulation of key traits in diverse crops.     

TILLING在水稻育种中的应用前景

TILLING(Targeting Induced Local Lesions in Genomes)是功能基因组研究中应用的一种反向遗传学技术。它能高通量低成本地在EMS诱变群体中鉴定出发生在特定基因上的点突变。在其基础上发展出的EcoTILLING技术则可发现种质资源中的SNP位点及小插入或缺失多态性位点。水稻是非常重要的粮食作物, 也是已经完成了全基因组序列测定,有丰富的生物信息学资源可以利用的基因组研究模式植物。水稻的分子标记辅助育种将在育种中扮演越来越重要的角色。在这样的背景下,本文从基于特定基因的种质资源鉴定、EMS诱变育种、及水稻功能标记开发等方面论述了其在水稻育种中的应用前景。     

Establishment of an in vitro fertilization system in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15–24 h after fusion, and developed into a globular-like embryo consisting of an average of 15–16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.     

Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa)

A simple mechanical method has been developed which allows the routine isolation of unfertilized and fertilized egg cells from ovules of Japonica and Indica rice varieties. In the experiments described, the majority of the egg cells and zygotes survived the isolation procedure when the donor plants were in a vigorous state. About 40% of the surviving zygotes underwent sustained development when cultured in Millicell inserts with a non-morphogenic rice feeder-cell culture. Nearly all zygote-derived callus cultures regenerated multiple shoots, which could be subsequently rooted with high efficiency. Zygote-derived plantlets matured to fertile plants when transplanted to soil. So far, about 80 independent plants each from the Japonica variety 'Taipei309' and the Indica variety 'IR58' have been regenerated. The potential of this single-cell regeneration system for marker gene-free transformation is discussed. Received: 26 November 1998 / Revision received: 15 March 1999 / Accepted: 21 March 1999     

Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes(TILLING), which combines traditional chemical mutagenesis with high‐throughput genome‐wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or evenallohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small‐scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2(or M3) plants.     

High throughput SNP genotyping with two mini-sequencing assays

Single nucleotide polymorphisms (SNPs) are veryimportant markers that can be used in many areas such asevolutionary genetics [1], disease-susceptibility genes[2,3], personalized medicine and forensics. Only about20% of human polymorphisms are length polymorphisms,whereas about 80% of human polymorphisms areSNPs. Kruglyak et al. [4] reported that there were about11,000,000 SNPs in the world population. There are many kinds of SNP genotyping technology[5,6]: some are only suitable to low …     

水稻“双－3”多胚发生的研究

水稻ＭＩＶ（双－３、籼稻）传粉后可以有多个花粉管同时进入胚囊．大多数胚囊的合子发育为一个正常的胚,但是有少数合子胚发生裂生并分化形成双胚芽和一胚根．有些胚囊的助细胞和卵细胞同时受精后,分别发育为助细胞胚和合子胚;有些胚囊中的反足细胞团可直接发育为胚．可见“双－３”水稻除有正常合子胚外还存在助细胞胚和反足细胞匹的多胚现象．     

水稻“双－3”多胚发生的研究

叶秀,陈泽濂,黎垣庆 　　水稻ＭＩＶ（双－３、籼稻）传粉后可以有多个花粉管同时进入胚囊．大多数胚囊的合子发育为一个正常的胚,但是有少数合子胚发生裂生并分化形成双胚芽和一胚根．有些胚囊的助细胞和卵细胞同时受精后,分别发育为助细胞胚和合子胚;有些胚囊中的反足细胞团可直接发育为胚．可见“双－３”水稻除有正常合子胚外还存在助细胞胚和反足细胞匹的多胚现象．     

DEco‐TILLING: an inexpensive method for single nucleotide polymorphism discovery that reduces ascertainment bias

Genetic markers that measure DNA variation are important for population genetics research, resource management and other applications. Single nucleotide polymorphisms (SNP) are becoming a popular marker because they are abundant and because rapid and efficient assays can be developed to detect them. However, with the exception of a few organisms, most species have little DNA sequence information available and relatively few SNPs have been developed for species that lack sequence data. Methods to find SNPs can be expensive and incorporate substantial ascertainment bias, which may result in failure to discover SNPs that are useful or efficient for addressing specific questions. We have developed a system to detect SNPs that we call DEco‐TILLING, which is derived from Eco‐TILLING (targeting induced local lesions in genomes). The DEco‐TILLING method facilitates the development of useful genotyping assays rapidly and inexpensively and can reduce ascertainment bias.     

TILLING技术在植物功能基因组及育种中的应用

随着拟南芥、水稻等模式植物基因组测序计划的全面完成,数据库中大量的DNA序列需要进行功能注释,而用传统的正向遗传学进行基因克隆和近年来发展的反向遗传学（如插入突变、反义RNA、RNAi等技术）方法已不能适应基因组学的发展需求,因此,研发大规模、高通量的基因功能分析方法成为当务之急。TILLING技术（Targeting induced local lesions in genomes）就是在基因组生物学大背景下出现的一种全新的反向遗传学技术。TILLING技术的基本步骤是通过化学诱变方法产生一系列点突变,经过PCR扩增放大和变性复性过程产生异源双链DNA分子,再通过特异性酶切和双色电泳分析识别异源双链中错配碱基,从而检测出突变发生的准确位置。由于具有高通量、大规模、高灵敏度和自动化等特点,能够适应植物功能基因组学研究的要求,TILLING技术已经和即将在功能基因组领域发挥越来越重要的作用。TILLING技术应用于已测序完成的拟南芥和水稻中的突变位点检测并取得了巨大成功;TILLING技术应用于农作物的品种改良,可以帮助实现快速、定向改良作物的品种,同时由于TILLING采用的化学诱变技术与传统诱变育种并无二致,因此在作物改良中采用TILLING技术不存在外源基因转入引发的转基因作物（GMO）争论;由TILLING技术发展来的EcoTILLING技术,具有通量高、成本低、定位准确等优点,可以很好地进行多态性检测和研究基因的功能,已成为开展物种DNA多态性检测和不同物种演替进化研究的有力工具。本文简要介绍了TILLING的原理及操作步骤,讨论了TILLING技术的特点和优点及TILLING技术的应用前景。     

An ultraperformance liquid chromatography method for the normal-phase separation of lipids

An ultraperformance liquid chromatography method using normal-phase solvents, a silica column, and evaporative light-scattering detection is presented. The method is based on a quaternary gradient profile and is capable of resolving the major neutral and polar lipids present in plasma and animal tissue in under 5 min, with a total cycle time of 11 min. Limits of quantitation for 7 different lipid classes were on the order of 200 ng of material on column which enables an accurate analysis from as little as 20 μL of plasma or 50 mg of tissue for typical samples. Intraday and interday precision for the determination of the major lipid classes in human plasma ranged from 3.6 to 10.5% CV with a variability in retention time of less than 6%. The utility of the method is demonstrated through the separation and quantitation of lipids in mouse plasma, liver, and heart tissue.     

水稻卵细胞与合子的分离

水稻卵细胞与合子的分离韩红梅赵洁施华中杨弘远周嫦（武汉大学生命科学学院植物生殖生物学研究室武汉４３００７２）关键词水稻,卵细胞,合子,分离ＩＳＯＬＡＴＩＯＮＯＦＥＧＧＣＥＬＬＳＡＮＤＺＹＧＯＴＥＳＩＮＯＲＹＺＡＳＡＴＩＶＡＨＡＮＨｏｎｇＭｅｉＺ...     

牵牛花受精前后卵细胞质体和线粒体及其细胞质DNA的变化

应用透射电镜研究了圆叶牵牛（Ｐｈａｒｂｉｔｉｓｐｕｒｐｕｒｅａ （Ｌ．） Ｖｏｙｇｈｔ）和大花牵牛（Ｐ． ｌｉｍ ｂａｔａ Ｌｉｎｄｌ．）卵细胞在受精前和受精后质体和线粒体的变化。观察到卵细胞在受精前高度液泡化,在核周及卵细胞周围的薄层细胞质中有质体和线粒体。质体含１～２ 个较大淀粉粒。线粒体多呈环形或杯状。卵细胞受精后,细胞质增多,质体的数量也明显增加。质体基质变得更加浓厚,并且普遍含有嗜锇的球体。合子中线粒体丰富,但缺乏卵细胞中那种环形或杯状线粒体,多呈圆形。细胞质ＤＮＡ检测的结果表明,卵细胞质ＤＮＡ荧光有大小和形状不同的两类,它们在细胞中随机分布。一类较大,呈环状,可能是线粒体中的ＤＮＡ显示的荧光;另一类小的点状荧光,可能大多是质体ＤＮＡ荧光,前者比后者多。卵细胞受精后,细胞质类核发生变化,表现在数量上明显地比受精前少。研究揭示了牵牛花合子中细胞质ＤＮＡ 减少的现象,说明了母系质体ＤＮＡ 在受精后可能被降解,提供了母系质体不传递到后代的可能机制。     

Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis

Background

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.

Results

Here we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified.

Conclusions

Using this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1263-4) contains supplementary material, which is available to authorized users.