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A TRIM Insertion in the Promoter of Ms2 Causes Male Sterility in Wheat

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A TRIM Insertion in the Promoter of Ms2 Causes Male Sterility in Wheat

Chuan Xia et al. Nat Commun.

Abstract

The male-sterile ms2 mutant has been known for 40 years and has become extremely important in the commercial production of wheat. However, the gene responsible for this phenotype has remained unknown. Here we report the map-based cloning of the Ms2 gene. The Ms2 locus is remarkable in several ways that have implications in basic biology. Beyond having no functional annotation, barely detectable transcription in fertile wild-type wheat plants, and accumulated destructive mutations in Ms2 orthologs, the Ms2 allele in the ms2 mutant has acquired a terminal-repeat retrotransposon in miniature (TRIM) element in its promoter. This TRIM element is responsible for the anther-specific Ms2 activation that confers male sterility. The identification of Ms2 not only unravels the genetic basis of a historically important breeding trait, but also shows an example of how a TRIM element insertion near a gene can contribute to genetic novelty and phenotypic plasticity.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Map-based cloning and characterization of Ms2.
Phenotypes of floral organs, following the removal of the palea and lemma, of Chinese Spring (a) and ms2 mutant plants (b). The anther structure of wild type at the pachytene stage (c), metaphase stage (e) and telophase stage (g); ms2 anther at the pachytene stage (d), metaphase stage (f) and telophase stage (h). (i) The region of Brachypodium distachyon chromosome 4 containing 11 genes syntenic with the Ms2 region of wheat chromosome 4D (j), with orthologs indicated by diagonal lines. Vertical lines indicate mapping marker locations, with the number of recombinant chromosomes (R) noted underneath. Gene no.5 (orange) is PAMs2, and gene no. 14 (red) is Ms2. The blue triangle indicates the TRIM insertion. (k) Gene structure of Ms2. Black boxes indicate exons and the SNP positions of five EMS mutant lines are indicated and labelled. The blue boxes indicate the TRIM insertion. Bars=500 μm in a,b, and 50 μm in ch.
Figure 2
Figure 2. Transgenic wheat plants, EMS mutants and gene expression analysis.
(a) The RT–PCR result of Ms2 expression in three reproductive organs (spikelets, emasculated spikelets, anthers) of wild type (Ms2-TRIM, fertile) and ms2 mutant (Ms2-TRIM+, sterile) plants in the Yanzhan1 genetic background. Phenotypes of floral organs, following the removal of the palea and lemma, of different wheat plants. (b) wheat cultivar ‘Fielder' transformed with empty vector. (c) wheat cultivar ‘Fielder' transformed with the Ms2-TRIM+Pro: Ms2 construct. (d) EMS-induced Ai-Bai (Rht-D1c/Ms2-TRIM+) mutant line m5. In situ hybridization analysis (antisense probe) of Ms2 in cross-sections of prophase stage anthers from wild type (e) and ms2 mutant (f) plants. Bars=500 μm in bd, and 50 μm in e,f.
Figure 3
Figure 3. Predicted gene structures of Ms2 and haplotype analysis of the coding sequence of Ms2.
(a) Predicted gene structures of the Ms2 loci in the A, B and D diploid ancestor genomes (left panel) and the A, B and D subgenomes of hexaploid wheat (right panel). ORFs are depicted as coloured boxes; the truncated portions of ORFs are depicted as white boxes; red arrows denote the positions of premature stop codons. The orange triangle indicates an insertion in the third exon in the A diploid ancestor genome and in the A subgenome. Exons missing in the B diploid ancestor genome are indicated with dashed boxes. Double arrows in dark blue denote the 17-bp insertion in exon 7. (b) Haplotype analysis of the coding sequence of Ms2. The DNA polymorphisms in the coding sequence are listed at the top. Each column corresponds to a polymorphic site (SNP/Indel), and each row indicates one haplotype of Ms2. The horizontal stacked bar plot indicates, in sequence, the percentage of each haplotype among the wild Ae. tauschii (diploid D) accessions, the hexaploid landraces and the modern wheat varieties. The Chinese Spring (hexaploid 4D) allele of Ms2 was used as the reference (listed in the last row) and is indicated by light yellow cells; blue cells indicate alternate alleles; rust red cells indicate an allele with a premature stop codon. (c) Coding sequences at the end of exon 7 and exon 8 that distinguish ORF-I, ORF-II and ORF-III. The left panel indicates the proposed evolution trajectory of the Ms2 allele in the ms2 mutant.
Figure 4
Figure 4. The proposed evolutionary trajectory of Ms2 and a model explaining the fertile and male-sterile phenotypes of different genotypes.
In the A and B diploid ancestor genomes, Ms2 othorlogs each accumulated one deleterious mutation. Three possible ORFs were predicted for the D diploid ancestor genome. ORF-I was not detected in hexaploidy wheat. In the ms2 mutant and in the transgenic Ms2-TRIM+wheat (Fielder background), Ms2 (ORF-III) is activated by the TRIM insertion, leading to male sterility. The EMS-mutagenized mutant lines, which have missense mutations in Ms2, produced normal or larger-than-mutant anthers. The purple arrow indicates Ms2, the gray arrows indicate truncated ORFs, the yellow arrow indicates the new ORF derived from the frame shift and the subsequent loss of the stop codon. The red triangles indicate the TRIM element in the promoter of Ms2.

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