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, 8 (7), e68529

MutMap+: Genetic Mapping and Mutant Identification Without Crossing in Rice

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MutMap+: Genetic Mapping and Mutant Identification Without Crossing in Rice

Rym Fekih et al. PLoS One.

Abstract

Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences. We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line. Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual. Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line. This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing. Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A simplified scheme of MutMap+.
(A) Seeds harvested following EMS mutagenesis of rice at immature embryo stage are used to establish M1 generation, at which stage most of mutations incorporated by EMS are in the heterozygous state. (B) M2 progeny obtained from a self-fertilized M1 plant segregate for wild-type (indicated by green color) and mutant (brown color) phenotypes. Here we focus on wild-type heterozygous individuals. (C) Heterozygous M2 plant are selfed to obtain M3 progeny that segregate 3∶1 for wild-type and mutant phenotypes. Genomic DNA from 20–40 M3 mutant and wild-type M3 progeny are separately bulked, and subjected to whole-genome sequencing. The resulting short reads are aligned to reference sequence of the cultivar used for mutagenesis. (D) SNP-index is calculated for each SNP, and plots relating SNP-index and chromosome positions are obtained for both the mutant and wild-type M3 bulks separately. The two SNP-index plots are compared to identify the region with SNP-index = 1 that is specific to the mutant bulk. (E) We can also evaluate Δ(SNP-index) plot, which is obtained by subtracting SNP-index value of wild-type bulk from that of mutant bulk. Genomic region harboring the causal mutation should have positive Δ(SNP-index) values.
Figure 2
Figure 2. MutMap+ identifies the causal mutation of the Hit9188 early stage lethality phenotype.
(A) The Hit9188 mutant is characterized by seedlings with pale-green and dwarf phenotypes that eventually die out starting from about three weeks after germination. (B) Chromosome 1 SNP-index plot for mutant and wild-type (WT) bulks derived from a segregating Hit9188 M3 progeny that was obtained by selfing a heterozygous M2 plant, as well as the Δ(SNP-index) plot generated by subtraction of WT bulk from mutant bulk. Points in the graphs represent SNPs, and the red lines represent the sliding window average of 4 Mb interval with 10 Kb increment. Shaded areas correspond to the genomic region where SNP-indices of mutant bulk and wild-type bulk show statistically significant (P<0.05) differences (i.e. Δ(SNP-index) >0). (C) Genomic location and structure of the Os01g0127300 gene harboring the Hit9188 mutant candidate nucleotide change, whose position within the gene together with the predicted amino acid change are indicated by a red triangle. (D) The deduced amino acid sequence of the protein encoded by Os01g0127300. The red font indicates the mutated alanine residue. (E) DNA sequencing peak chromatograms of the region spanning the Os01g0127300 mutation showing the wild-type G in Hitomebore, mutant A in mutant bulk, and the heterozygous G/A in wild-type (WT) bulk.
Figure 3
Figure 3. RNA interference confirms that the Os01g0127300 mutation is responsible for Hit9188 developmental phenotypes.
(A) Structure of Os01g0127300 (OsNAP6) and scheme of the construct used for RNAi analysis targeting the OsNAP6 gene. (B) Results of real-time quantitative reverse transcription (RT)-PCR showing the relative expression level of OsNAP6 in rice plants transformed with OsNAP6-RNAi construct (RNAi) and empty vector (Empty). Asterisks indicate significant differences (Student’s t-test, **P<0.01). (C) Phenotype of leaf blade (top) and seedlings (bottom) of OsNAP6 RNAi transgenic plants compared to the Hit9188 mutant and Hitomebore wild-type (WT) plants.
Figure 4
Figure 4. MutMap+ identifies the genomic region harboring the Hit11440 mutation.
(A) Phenotype of wild-type (WT) and Hit11440 mutant seedlings at about 12 days after sowing. (B) Chromosome 8 SNP-index plots for mutant and wild-type (WT) bulks derived from DNA bulks of M3 progeny obtained from selfing of a heterozygous M2 plant, and the Δ(SNP-index) plot generated by subtracting the WT bulk SNP-indices from that of the mutant bulk. Points in the graphs represent SNPs, and red lines represent the sliding window average of 4 Mb interval with 10 Kb increment. Shaded areas correspond to the genomic region where SNP-indices of WT and mutant bulks show statistically significant (P<0.05) differences (i.e. Δ(SNP-index) >0). (C) Genomic location and structure of the candidate gene, Os08g0139100, harboring a nucleotide change in the Hit11440 mutant. Location of the mutated glutamine residue is indicted by a red triangle. (D) The predicted 299-amino acid sequence of the DAG protein encoded by Os08g0139100. The mutated glutamine (Q) residue is indicated in red. (E) Sanger sequencing confirms the candidate SNP identified by Illumina whole genome re-sequencing as indicated by peak chromatograms of the region spanning the Os08g0139100 mutation. The wild-type C in Hitomebore, the mutated T in mutant-bulk DNA, and the C/T mixture in wild-type (WT) bulk DNA are indicted by the black arrows.

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Grant support

This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation PMI-0010) and Grant-in-aid for Scientific Research from the Ministry of Education, Cultures, Sports and Technology, Japan, to HS and RT (Grant-in-Aid for Scientific Research on Innovative Areas 23113009) and JSPS KAKENHI to RT (Grant No. 24248004). RF is a postdoctoral fellow of the Japanese Society for the Promotion of Sciences (JSPS). LC and S. Kamoun are supported by the Gatsby Charitable Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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