The fruits of diploid and octoploid strawberry (Fragaria spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in MYB10 cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F. 3ananassa). Using a mapping-by-sequencing approach, we identified a gypsytransposon in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional loss-of-function mutations in MYB10 were identified among geographically diverse white-fruited F. vesca ecotypes. Genetic and transcriptomic analyses of octoploid Fragaria spp revealed that FaMYB10-2, one of three MYB10 homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in FaEnSpm-2 are responsible for enhanced MYB10-2 expression and anthocyanin biosynthesis in strawberry fruit flesh.

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Background NAC transcription factors contain five highly conserved subdomains which are required for protein dimerisation and DNA binding. Few residues within these subdomains have been identified as essential for protein function, and fewer still have been shown to be of biological relevance in planta. Here we use a positive regulator of senescence in wheat, NAM-A1, to test the impact of missense mutations at specific, highly conserved residues of the NAC domain on protein function. Results We identified missense mutations in five highly conserved residues of the NAC domain of NAM-A1 in a tetraploid TILLING population. TILLING lines containing these mutations, alongside synonymous and non-conserved mutation controls, were grown under glasshouse conditions and scored for senescence. Four of the five mutations showed a significant and consistent delay in peduncle senescence but had no consistent effects on flag leaf senescence. All four mutant alleles with the delayed senescence phenotype also lost the ability to interact with the homoeolog NAM-B1 in a yeast two-hybrid assay. Two of these residues were previously shown to be involved in NAC domain function in Arabidopsis, suggesting conservation of residue function between species. Three of these four alleles led to an attenuated cell death response compared to wild-type NAM-A1 when transiently over-expressed in Nicotiana benthamiana. One of these mutations was further tested under field conditions, in which there was a significant and consistent delay in both peduncle and leaf senescence. Conclusions We combined field and glasshouse studies of a series of mutant alleles with biochemical analyses to identify four residues of the NAC domain which are required for NAM-A1 function and protein interaction. We show that mutations in these residues lead to a gradient of phenotypes, raising the possibility of developing allelic series of mutations for traits of agronomic importance. We also show that mutations in NAM-A1 more severely impact peduncle senescence, compared to the more commonly studied flag leaf senescence, highlighting this as an area deserving of further study. The results from this integrated approach provide strong evidence that conserved residues within the functional domains of NAC transcription factors have biological significance in planta.

Durum wheat ( Triticum turgidum ) derives from a hybridization event approximately 400,000 years ago which led to the creation of an allotetraploid genome. The evolutionary recent origin of durum wheat means that its genome has not yet been fully diploidised. As a result, many of the genes present in the durum genome act in a redundant fashion, where loss-of-function mutations must be present in both gene copies to observe a phenotypic effect. Here, we use a novel set of induced variation within the cv. Kronos TILLING population to identify a locus controlling a dominant, environmentally dependent chlorosis phenotype. We carried out a forward screen of the sequenced cv. Kronos TILLING lines for senescence phenotypes and identified a line with a dominant early senescence and chlorosis phenotype. Mutant plants contained less chlorophyll throughout their development and displayed premature flag leaf senescence. A segregating population was classified into discrete phenotypic groups and subjected to bulked-segregant analysis using exome capture followed by next-generation sequencing. This allowed the identification of a single region on chromosome 3A, Yellow Early Senescence 1 ( YES-1 ), which was associated with the mutant phenotype. While this phenotype was consistent across 4 years of field trials in the United Kingdom, the mutant phenotype was not observed when grown in Davis, CA (United States). To obtain further SNPs for fine-mapping, we isolated chromosome 3A using flow sorting and sequenced the entire chromosome. By mapping these reads against both the cv. Chinese Spring reference sequence and the cv. Kronos assembly, we could identify high-quality, novel EMS-induced SNPs in non-coding regions within YES-1 that were previously missed in the exome capture data. This allowed us to fine-map YES-1 to 4.3 Mb, containing 59 genes. Our study shows that populations containing induced variation can be sources of novel dominant variation in polyploid crop species, highlighting their importance in future genetic screens. We also demonstrate the value of using cultivar-specific genome assemblies alongside the gold-standard reference genomes particularly when working with non-coding regions of the genome. Further fine-mapping of the YES-1 locus will be pursued to identify the causal SNP underpinning this dominant, environmentally dependent phenotype.

During the last two decades, new virulent and aggressive races of Puccinia striiformis Westend. f. sp. tritici (Pst) have spread worldwide, causing devastating epidemics and prompting the search for new sources of resistance in wheat (Triticum aestivum L.). Between 2012 and 2017, we mapped four stripe rust resistance quantitative trait loci (QTL) effective against the Pst races present in California, USA, using recombinant inbred lines (RILs) developed from the cross between the Argentinean cultivars ‘Klein Proteo’ and ‘Klein Chajá’. The RIL population showed transgressive segregation in all six growing seasons relative to the parental lines, which showed moderate levels of Pst resistance. Analyses by year detected QTL conferring adult plant resistance on chromosomes 1BL, 2BS, 3D centromeric (from Klein Chajá), and 4DL (from Klein Proteo). QYr.ucw‐1BL, mapped in the Yr29 resistance gene region, was significant in all seasons (P < 0.01) and explained on average 31.0 to 32.8% of the observed variation. QYr.ucw‐2BS showed a stronger effect than QYr.ucw‐1BL in 2013 but was ineffective in 2014 and 2016. This QTL also conferred seedling resistance, suggesting that it is an all‐stage resistance gene. Centromeric QYr.ucw‐3D and QYr.ucw‐4DL showed smaller effects than the previous QTL and were significant only in some of the experiments. No significant interactions were detected among QTL, indicating the absence of digenic epistatic effects. The molecular markers identified in this study can be used to combine these genes and accelerate their deployment in wheat breeding programs.

Key Message Several Fusarium wilt resistance genes were discovered, genetically and physically mapped, and rapidly deployed via marker-assisted selection to develop cultivars resistant toFusarium oxysporumf. sp.fragariae, a devastating soil-borne pathogen of strawberry. Abstract Fusarium wilt, a soilborne disease caused by Fusarium oxysporum f. sp. fragariae, poses a significant threat to strawberry (Fragaria$$\times$$ × ananassa) production in many parts of the world. This pathogen causes wilting, collapse, and death in susceptible genotypes. We previously identified a dominant gene (FW1) on chromosome 2B that confers resistance to race 1 of the pathogen, and hypothesized that gene-for-gene resistance to Fusarium wilt was widespread in strawberry. To explore this, a genetically diverse collection of heirloom and modern cultivars and octoploid ecotypes were screened for resistance to Fusarium wilt races 1 and 2. Here, we show that resistance to both races is widespread in natural and domesticated populations and that resistance to race 1 is conferred by partially to completely dominant alleles among loci (FW1, FW2, FW3, FW4, and FW5) found on three non-homoeologous chromosomes (1A, 2B, and 6B). The underlying genes have not yet been cloned and functionally characterized; however, plausible candidates were identified that encode pattern recognition receptors or other proteins known to confer gene-for-gene resistance in plants. High-throughput genotyping assays for SNPs in linkage disequilibrium with FW1-FW5 were developed to facilitate marker-assisted selection and accelerate the development of race 1 resistant cultivars. This study laid the foundation for identifying the genes encoded by FW1-FW5, in addition to exploring the genetics of resistance to race 2 and other races of the pathogen, as a precaution to averting a Fusarium wilt pandemic.

Core Ideas The adult‐plant stripe rust resistance locus QYr.ucw‐1BL is effective against new virulent races of the stripe rust pathogen. QYr.ucw‐1BL was mapped to a 332‐kb region including 13 genes associated with disease resistance. QYr.ucw‐1BL and the durable stripe rust resistance gene Yr29 map to the same region and confer similar resistance reactions. Exome capture data were useful for accelerating marker development and establishing haplotypes associated with resistant and susceptible lines. High‐throughput molecular markers have been developed to accelerate the deployment of QYr.ucw‐1BL and the durable stripe rust resistance gene Yr29. The appearance of highly virulent and more aggressive races of Puccinia striiformis f. sp. tritici (Pst) during the last two decades has led to stripe rust epidemics worldwide and to the rapid erosion of effective resistance genes. In this study, we mapped an adult‐plant resistance locus from the Argentinean wheat (Triticum aestivum L.) cultivar Klein Chajá, which is effective against these new Pst races. By using wheat exome capture data and a large population of 2480 segregating plants (4960 gametes), we mapped QYr.ucw‐1BL within a 0.24‐cM region [332 kb in International Wheat Genome Sequencing Consortium (IWGSC) RefSeq version 1.0] on chromosome arm 1BL. This region overlaps with current maps of the adult‐plant Pst resistance gene Yr29, which has remained effective for more than 60 yr. An allelism test failed to find recombination between QYr.ucw‐1BL and Yr29 and yielded similar resistance phenotypes for the two loci. These results, together with similar haplotypes in the candidate region, suggested that QYr.ucw‐1BL and Yr29 might represent the same gene. However, we cannot rule out the possibility of tightly linked but different genes because most of the 13 genes in the candidate region are annotated with functions associated with disease resistance. To evaluate their potential as QYr.ucw‐1BL/Yr29 candidate genes, we characterized their polymorphisms between resistant and susceptible haplotypes. Finally, we used these polymorphisms to develop high‐throughput markers to accelerate the deployment of these Pst resistance loci in wheat breeding programs.

Anthocyanins are the principal color-producing compounds synthesized in developing fruits of strawberry (Fragaria spp.). Substantial natural variation in color have been observed in fruits of diploid and octoploid accessions, resulting from distinct accumulation and distribution of anthocyanins in fruits. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 has been shown as the main activator in strawberry fruit. Here, we show that MYB10 mutations cause most of the anthocyanin variation observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F. ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsytransposon insertion in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional lossof-function MYB10 mutations were identified among geographically diverse whitefruited F. vesca ecotypes. Genetic and transcriptomic analyses in octoploid Fragaria spp. revealed that FaMYB10-2, one of three MYB10 homoeologs identified, residing in the F. iinumae-derived subgenome, regulates the biosynthesis of anthocyanins in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression and anthocyanin accumulation. Our findings suggest that putative cis regulatory elements provided by FaEnSpm-2 are required for high and ectopic MYB10-2 expression and induction of anthocyanin biosynthesis in fruit flesh. We developed MYB10-2 (sub-genome) specific DNA markers for marker-assisted selection that accurately predicted anthocyanin phenotypes in octoploid segregating populations.