Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

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 F2 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. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009; Irvine et al. 2009; Schneeberger et al. 2009; Zuryn et al. 2010; Austin et al. 2011). However, current methods depend on identifying homozygous mutant individuals in an F2 mapping population after outcrossing (Schneeberger et al. 2009; Austin et al. 2011) or require several rounds of backcrossing (Zuryn et al. 2010), a timeconsuming requirement not easily met in organisms with long generation times.

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 F1 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 SRM by cloning a gametophyte lethal mutation in Arabidopsis thaliana, for which the recovery of homozygotes is not possible.

As proof of principle, we aimed to map the gene affected in a pollen-tube reception mutant obtained from a forward genetic screen using EMS-treated seeds of A. thaliana (Col-0 accession, Supporting Information, File S1). The turan-1 (tun-1) mutant disrupts cell-cell communication between male and female gametophytes, which is indispensable for fertilization. In flowering plants, the gametes are produced by the haploid, multicellular gametophytes. The male gametophyte (pollen tube) delivers two sperm cells to the female gametophyte (embryo sac), harboring two female gametes. Fertilization of the egg and central cell forms the embryo and the endosperm, respectively. In heterozygous tun-1 mutants, 12% (n = 1318 ovules) of the embryo sacs remain unfertilized, compared to only 1. …

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