SNP-Ratio Mapping (SRM): Identifying Lethal Alleles and Mutations in Complex Genetic Backgrounds by Next-Generation Sequencing

Abstract

We present a generally applicable method allowing rapid identification of causal alleles in mutagenized genomes by nextgeneration sequencing. Currently used approaches rely on recovering homozygotes or extensive backcrossing. In contrast, SNP-ratio mapping allows rapid cloning of lethal and/or poorly transmitted mutations and second-site modifiers, which are often in complex genetic/transgenic backgrounds. FORWARD genetic screens are powerful in uncovering novel gene functions in genetic model organisms. While some mutant screens can be quick to perform, the identification of the causative mutation by map-based cloning is extremely labor-intensive. Large F 2 mapping populations of .1000 mutant individuals are required (Lukowitz et al. 2000;Jander et al. 2002) to fine-map a chromosomal region harboring a causative mutation. This number of mutant individuals can be difficult to obtain, especially when working with phenotypic traits that (i) are difficult to score, (ii) are weakly transmitted, or (iii) are in organisms that are hard to propagate. The recent development of next-generation sequencing (NGS) platforms has made sequencing of whole genomes quick and affordable. One application of NGS is to replace map-based cloning by the sequencing of mutagenized genomes to quickly identify causative mutations, a method successfully applied in many model organisms (Sarin et al. Here, we describe a generally applicable method, SNPratio mapping (SRM), which allows the rapid identification of lethal and/or poorly transmitted mutations and secondsite modifiers by NGS. It is based on the distinct segregation ratio of the causative (and linked) single-nucleotide polymorphism(s) (SNPs) from that of unlinked SNPs. SRM allows the mapping of lethal mutations after only two rounds of backcrossing via NGS. After backcrossing twice to the non-mutagenized parent, any unlinked SNP created by ethyl methanesulfonate (EMS) mutagenesis segregates 1:3 in a pool of individuals. By selecting only mutant individuals in the F 1 generation of the second backcross (BC2), the causative SNP is enriched and segregates 1:1 in a pool of mutant BC2 individuals (Figure 1). Thus, calculating the SNP/non-SNP segregation ratio allows the quick identification of the causative mutation. The method is applicable to any model organism and mutagen causing mostly point mutations or small indels. SRM is the method of choice when working with (i) lethal mutations, (ii) hard-to-score phenotypes, (iii) mutations with low transmission, and (iv) second-site modifiers in complex genetic/ transgenic backgrounds. Here, we demonstrate the power of

Receptor-like cytoplasmic kinase MARIS functions downstream of Cr RLK1L-dependent signaling during tip growth

“…Briefly, the SNPs called with Freebayes were fed to a hidden Markov chain-based algorithm that estimates the probability that the SNP is the causative SNP, based on the read data and linkage between the SNPs within each chromosome. For mri-3D/MRI, the top candidate exonic SNP had a ratio of alternate to reference reads of 0.476, very close to the theoretically expected ratio of 0.500 (Datasets S1 and S2) (24). The affected gene AT2G41970 encodes an uncharacterized RLCK, henceforth referred to as "MARIS" (MRI) (see below).…”

“…1B). To identify the EMS-generated causative lesion in mri-3D, likely a single nucleotide polymorphism (SNP), an SNP-ratio mapping (SRM) approach (24), was carried out with modifications (SI Material and Methods). Briefly, the SNPs called with Freebayes were fed to a hidden Markov chain-based algorithm that estimates the probability that the SNP is the causative SNP, based on the read data and linkage between the SNPs within each chromosome.…”

Next-Generation Sequencing-Based Approaches for Mutation Mapping and Identification inCaenorhabditis elegans

“…Conversely, when using reverse VDM, the ratios of parental alleles are zero within the mapping region. An alternative strategy has been demonstrated that depends on backcrossing twice to the nonmutagenized starting strain and then selecting heterozygous mutant animals with the dominant phenotype for sequencing (Lindner et al 2012). Allele frequency will be 0.5 for linked alleles, and 0.25 for unlinked alleles, and this can be detected by plotting allele frequencies.…”

“…In a proof-of-principle study, it has been shown that significant library bias is not introduced when starting with low genome DNA input, and comparable mapping and variant detection results were obtained (Smith et al 2016); 50 hand-picked sterile F 2 recombinants yielded enough DNA for library construction. If it is possible to directly identify heterozygous F 2 animals unambiguously or by assessing F 3 phenotypes, then the double backcross method mentioned above (see previous section 4.7.1) could in principle also be used (Lindner et al 2012).…”

Growth Control: A Saga of Cell Walls, ROS, and Peptide Receptors

“…Remarkably, NTA becomes polarly localized in synergid cells upon pollen tube arrival, and this relocalization strictly depends on the presence of FER, suggesting that the two proteins act together in pollen tube reception, possibly in the same complex. Other mutants also cause a pollen tube phenotype similar to that of fer, such as mutants for the glycosyl-phosphatidylinositol-anchored protein LORELEI (LRE) and the UDP-glycosyltransferase TURAN (Capron et al, 2008;Tsukamoto et al, 2010;Lindner et al, 2012b). Like FER, LRE was shown to bind the small GTPase ROP2 and to be required for the production of female gametophytic ROS, suggesting that FER and LRE act in the same pathway presumably through a transient ROP2 signaling complex (Duan et al, 2014).…”