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.

Department of Plant Sciences University of California Davis Davis CA United States

Institute of Experimental Botany Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc Czechia

John Innes Centre Norwich United Kingdom

School of Biosciences University of Birmingham Birmingham United Kingdom

See more in PubMed

10+Wheat Genomes Project (2016). The Wheat ’Pan Genome’. Available at: http://www.10wheatgenomes.com/ (accessed January 25, 2019).

Acevedo-Garcia J., Spencer D., Thieron H., Reinstädler A., Hammond-Kosack K., Phillips A. L., et al. (2017). mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic Tilling approach. Plant Biotechnol. J. 15 367–378. 10.1111/pbi.12631 PubMed DOI PMC

Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215 403–410. 10.1006/jmbi.1990.9999 PubMed DOI

Avni R., Zhao R., Pearce S., Jun Y., Uauy C., Tabbita F., et al. (2014). Functional characterization of Gpc-1 genes in hexaploid wheat. Planta 239 313–324. 10.1007/s00425-013-1977-y PubMed DOI PMC

Borrill P., Adamski N., Uauy C. (2015). Genomics as the key to unlocking the polyploid potential of wheat. New Phytol. 208 1008–1022. 10.1111/nph.13533 PubMed DOI

Borrill P., Harrington S. A., Simmonds J., Uauy C. (2018). Identification of transcription factors regulating senescence in wheat through gene regulatory network modelling. bioRxiv 456749. 10.1104/pp.19.00380 PubMed DOI PMC

Borrill P., Harrington S. A., Uauy C. (2019). Applying the latest advances in genomics and phenomics for trait discovery in polyploid wheat. Plant J. 97 56–72. 10.1111/tpj.14150 PubMed DOI PMC

Borrill P., Ramirez-Gonzalez R., Uauy C. (2016). EXPVIP: a customizable RNA-seq data analysis and visualization platform. Plant Physiol. 170:2172. 10.1104/pp.15.01667 PubMed DOI PMC

Brinton J., Uauy C. (2019). A reductionist approach to dissecting grain weight and yield in wheat. J. Integr. Plant Biol. 61 337–358. 10.1111/jipb.12741 PubMed DOI PMC

Clark J. W., Donoghue P. C. J. (2018). Whole-genome duplication and plant macroevolution. Trends Plant Sci. 23 933–945. 10.1016/j.tplants.2018.07.006 PubMed DOI

Clavijo B. J., Garcia Accinelli G., Wright J., Heavens D., Barr K., Yanes L., et al. (2017a). W2rap: a pipeline for high quality, robust assemblies of large complex genomes from short read data. bioRxiv 110999.

Clavijo B. J., Venturini L., Schudoma C., Accinelli G. G., Kaithakottil G., Wright J., et al. (2017b). An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations. Genome Res. 27 885–896. 10.1101/gr.217117.116 PubMed DOI PMC

Dodsworth S., Chase M. W., Leitch A. R. (2016). Is post-polyploidization diploidization the key to the evolutionary success of angiosperms? Bot. J. Linn. Soc. 180 1–5. 10.1111/boj.12357 DOI

Doležel J., Vrána J., Šafář J., Bartoš J., Kubaláková M., Šimková H. (2012). Chromosomes in the flow to simplify genome analysis. Funct. Integr. Genomics 12 397–416. 10.1007/s10142-012-0293-0 PubMed DOI PMC

Dubcovsky J., Dvorak J. (2007). Genome plasticity a key factor in the success of polyploid wheat under domestication. Science 316:1862. 10.1126/science.1143986 PubMed DOI PMC

Fu D., Szücs P., Yan L., Helguera M., Skinner J. S., Von Zitzewitz J., et al. (2005). Large deletions within the first intron in Vrn-1 are associated with spring growth habit in barley and wheat. Mol. Genet. Genomics 273 54–65. 10.1007/s00438-004-1095-4 PubMed DOI

Garrison E., Marth G. (2012). Haplotype-based Variant Detection from Short-read Sequencing. Available: https://ui.adsabs.harvard.edu/abs/2012arXiv1207.3907G (accessed July 01, 2012).

Gebert M., Meschenmoser K., Svidová S., Weghuber J., Schweyen R., Eifler K., et al. (2009). A root-expressed magnesium transporter of the Mrs2/Mgt gene family in Arabidopsis thaliana allows for growth in Low-Mg2+ environments. Plant Cell 21:4018. 10.1105/tpc.109.070557 PubMed DOI PMC

Giorgi D., Farina A., Grosso V., Gennaro A., Ceoloni C., Lucretti S. (2013). FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS One 8:e57994. 10.1371/journal.pone.0057994 PubMed DOI PMC

Greenwood J. R., Finnegan E. J., Watanabe N., Trevaskis B., Swain S. M. (2017). New alleles of the wheat domestication gene Q reveal multiple roles in growth and reproductive development. Development 144:1959. 10.1242/dev.146407 PubMed DOI

Harrington S. A., Cobo N., Karafiátová M., DoleŽel J., Borrill P., Uauy C. (2019a). Identification of a dominant chlorosis phenotype through a forward screen of the Triticum turgidum cv. Kronos TILLING population. bioRxiv 622076. PubMed PMC

Harrington S. A., Overend L. E., Cobo N., Borrill P., Uauy C. (2019b). Conserved residues in the wheat (Triticum aestivum) Nam-A1 Nac domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence. bioRxiv 573881. PubMed PMC

Hoagland D. R., Arnon D. I. (1950). The Water-Culture Method for Growing Plants Without Soil, Vol. 347 Berkeley, CA: College of Agriculture, University of California, 32.

Huang X., Feng Q., Qian Q., Zhao Q., Wang L., Wang A., et al. (2009). High-throughput genotyping by whole-genome resequencing. Genome Res. 19 1068–1076. 10.1101/gr.089516.108 PubMed DOI PMC

International Wheat Genome Sequencing Consortium (2018). Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science 361:eaar7191. 10.1126/science.aar7191 PubMed DOI

Jupe F., Witek K., Verweij W., Sliwka J., Pritchard L., Etherington G. J., et al. (2013). Resistance gene enrichment sequencing (RenSeq) enables reannotation of THE NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations. Plant J. 76 530–544. 10.1111/tpj.12307 PubMed DOI PMC

Kang K., Kim Y.-S., Park S., Back K. (2009). Senescence-induced serotonin biosynthesis and its role in delaying senescence in rice leaves. Plant Physiol. 150 1380–1393. 10.1104/pp.109.138552 PubMed DOI PMC

Kim D., Langmead B., Salzberg S. L. (2015). HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12 357–360. 10.1038/nmeth.3317 PubMed DOI PMC

Kimura E., Bell J., Trostle C., Neely C., Drake D. (2016). Potential Causes of Yellowing During the Tillering Stage of Wheat in Texas. Texas A&M AgriLife Extension. Available: https://agrilifecdn.tamu.edu/texaslocalproduce-2/files/2018/07/Potential-Causes-of-Yellowing-During-the-Tillering-Stage-of-Wheat-in-Texas.pdf (accessed June 06, 2019).

Krasileva K. V., Vasquez-Gross H. A., Howell T., Bailey P., Paraiso F., Clissold L., et al. (2017). Uncovering hidden variation in polyploid wheat. Proc. Natl. Acad. Sci. U.S.A. 114:E913. 10.1073/pnas.1619268114 PubMed DOI PMC

Krzywinski M., Schein J., Birol İ, Connors J., Gascoyne R., Horsman D., et al. (2009). Circos: an information aesthetic for comparative genomics. Genome Res. 19 1639–1645. 10.1101/gr.092759.109 PubMed DOI PMC

Kubaláková M., Vrána J., Číhalíková J., Šimková H., Doležel J. (2002). Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 104 1362–1372. 10.1007/s00122-002-0888-2 PubMed DOI

Li H. (2013). Aligning Sequence Reads, Clone Sequences and Assembly Contigs with BWA-MEM. Available: https://ui.adsabs.harvard.edu/abs/2013arXiv1303.3997L (accessed March 01, 2013).

Maccaferri M., Harris N. S., Twardziok S. O., Pasam R. K., Gundlach H., Spannagl M., et al. (2019). Durum wheat genome highlights past domestication signatures and future improvement targets. Nat. Genet. 51 885–895. 10.1038/s41588-019-0381-3 PubMed DOI

Mamanova L., Coffey A. J., Scott C. E., Kozarewa I., Turner E. H., Kumar A., et al. (2010). Target-enrichment strategies for next-generation sequencing. Nat. Methods 7 111–118. 10.1038/nmeth.1419 PubMed DOI

Mo Y., Howell T., Vasquez-Gross H., De Haro L. A., Dubcovsky J., Pearce S. (2018). Mapping causal mutations by exome sequencing in a wheat Tilling population: a tall mutant case study. Mol. Genet. Genomics 293 463–477. 10.1007/s00438-017-1401-6 PubMed DOI PMC

NOAA National Centers For Environmental Information (2017). State of the Climate: National Climate Report for Annual 2017. Asheville, NC: National Centers for Environmental Information.

Paterson A. H., Wang X., Li J., Tang H. (2012). “Ancient and recent polyploidy in monocots,” in Polyploidy and Genome Evolution, eds Soltis P., Soltis D. E. (Heidelberg: Springer; ).

Payandeh J., Pfoh R., Pai E. F. (2013). The structure and regulation of magnesium selective ion channels. Biochim. Biophys. Acta 1828 2778–2792. 10.1016/j.bbamem.2013.08.002 PubMed DOI

Pearce S., Tabbita F., Cantu D., Buffalo V., Avni R., Vazquez-Gross H., et al. (2014). Regulation of Zn and Fe transporters by the Gpc1 gene during early wheat monocarpic senescence. BMC Plant Biol. 14:368. 10.1186/s12870-014-0368-2 PubMed DOI PMC

Peng J., Richards D. E., Hartley N. M., Murphy G. P., Devos K. M., Flintham J. E., et al. (1999). ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature 400 256–261. 10.1038/22307 PubMed DOI

R Core Team (2018). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing.

Ramírez-González R. H., Borrill P., Lang D., Harrington S. A., Brinton J., Venturini L., et al. (2018). The transcriptional landscape of polyploid wheat. Science 361:eaar6089. 10.1126/science.aar6089 PubMed DOI

Ramirez-Gonzalez R. H., Segovia V., Bird N., Fenwick P., Holdgate S., Berry S., et al. (2015a). Rna-Seq bulked segregant analysis enables the identification of high-resolution genetic markers for breeding in hexaploid wheat. Plant Biotechnol. J. 13 613–624. 10.1111/pbi.12281 PubMed DOI

Ramirez-Gonzalez R. H., Uauy C., Caccamo M. (2015b). PolyMarker: a fast polyploid primer design pipeline. Bioinformatics 31 2038–2039. 10.1093/bioinformatics/btv069 PubMed DOI PMC

Rodríguez-Leal D., Lemmon Z. H., Man J., Bartlett M. E., Lippman Z. B. (2017). Engineering quantitative trait variation for crop improvement by genome editing. Cell 171 470–480.e8. 10.1016/j.cell.2017.08.030 PubMed DOI

Šimková H., Svensson J. T., Condamine P., Hřibová E., Suchánková P., Bhat P. R., et al. (2008). Coupling amplified Dna from flow-sorted chromosomes to high-density Snp mapping in barley. BMC Genomics 9:294. 10.1186/1471-2164-9-294 PubMed DOI PMC

Simons K. J., Fellers J. P., Trick H. N., Zhang Z., Tai Y.-S., Gill B. S., et al. (2006). Molecular Characterization of the major wheat domestication gene Q. Genetics 172 547–555. 10.1534/genetics.105.044727 PubMed DOI PMC

Singh S., Giri M. K., Singh P. K., Siddiqui A., Nandi A. K. (2013). Down-regulation of OSSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants. J. Biosci. 38 583–592. 10.1007/s12038-013-9334-7 PubMed DOI

Snowball K., Robson A. D. (1991). Nutrient Deficiencies and Toxicities in Wheat: A Guide for Field Identification. Mexico City, MX: CIMMYT.

Soltis P. S., Soltis D. E. (2016). Ancient WGD events as drivers of key innovations in angiosperms. Curr. Opin. Plant Biol. 30 159–165. 10.1016/j.pbi.2016.03.015 PubMed DOI

Steuernagel B., Periyannan S. K., Hernández-Pinzón I., Witek K., Rouse M. N., Yu G., et al. (2016). Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture. Nat. Biotechnol. 34 652–655. 10.1038/nbt.3543 PubMed DOI

Uauy C. (2017). Wheat genomics comes of age. Curr. Opin. Plant Biol. 36 142–148. 10.1016/j.pbi.2017.01.007 PubMed DOI

Uauy C., Distelfeld A., Fahima T., Blechl A., Dubcovsky J. (2006). A NAC GENE regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314 1298–1301. 10.1126/science.1133649 PubMed DOI PMC

Uauy C., Wulff B. B. H., Dubcovsky J. (2017). Combining traditional mutagenesis with new high-throughput sequencing and genome editing to reveal hidden variation in polyploid wheat. Annu. Rev. Genet. 51 435–454. 10.1146/annurev-genet-120116-024533 PubMed DOI

Vrána J., Cápal P., Šimková H., Karafiátová M., Čížková J., Doležel J. (2016). Flow analysis and sorting of plant chromosomes. Curr. Protoc. Cytom. 78 5.3.1–5.3.43. 10.1002/cpcy.9 PubMed DOI

Vrána J., Kubaláková M., Šimková H., Číhalíková J., Lysák M. A., Doležel J. (2000). Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156 2033–2041. PubMed PMC

Vullo A., Allot A., Zadissia A., Yates A., Luciani A., Moore B., et al. (2017). Ensembl Genomes 2018: an integrated omics infrastructure for non-vertebrate species. Nucleic Acids Res. 46 D802–D808. 10.1093/nar/gkx1011 PubMed DOI PMC

Wang W., Simmonds J., Pan Q., Davidson D., He F., Battal A., et al. (2018). Gene editing and mutagenesis reveal inter-cultivar differences and additivity in the contribution of TAGW2 homoeologues to grain size and weight in wheat. Theor. Appl. Genet. 131 2463–2475. 10.1007/s00122-018-3166-7 PubMed DOI PMC

Wang Y., He Y., Yang M., He J., Xu P., Shao M., et al. (2016). Fine mapping of a dominant gene conferring chlorophyll-deficiency in Brassica napus. Sci. Rep. 6:31419. 10.1038/srep31419 PubMed DOI PMC

Warnes G. R., Bolker B., Bonebakker L., Gentleman R., Huber W., Liaw A., et al. (2019). gplots: Various R Programming Tools for Plotting Data. R package version 3.0.1.1.

Wellburn A. R. (1994). The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144 307–313. 10.1007/s11120-013-9834-1 PubMed DOI

Wickham H. (2016). ggplot2: Elegant Graphics for Data Analysis. New York, NY: Springer-Verlag.

Wickham H., François R., Henry L., Müller K. (2019). dplyr: A Grammar of Data Manipulation. R package version 0.8.0.1.

Wickham H., Henry L. (2018). tidyr: Easily Tidy Data with ‘spread()’ and ‘gather()’ Functions. R package version 0.8.2.

Wu H., Shi N., An X., Liu C., Fu H., Cao L., et al. (2018). Candidate genes for yellow leaf color in common wheat (Triticum aestivum L.) and major related metabolic pathways according to transcriptome profiling. Int. J. Mol. Sci. 19:1594. 10.3390/ijms19061594 PubMed DOI PMC

Wysoker A., Handsaker B., Marth G., Abecasis G., Li H., Ruan J., et al. (2009). The sequence alignment/map format and samtools. Bioinformatics 25 2078–2079. 10.1093/bioinformatics/btp352 PubMed DOI PMC

Yan L., Helguera M., Kato K., Fukuyama S., Sherman J., Dubcovsky J. (2004). Allelic variation at THE VRN-1 PROMOTER region in polyploid wheat. Theor. Appl. Genet. 109 1677–1686. 10.1007/s00122-004-1796-4 PubMed DOI

Yan L., Loukoianov A., Tranquilli G., Helguera M., Fahima T., Dubcovsky J. (2003). Positional cloning of the wheat vernalization gene Vrn1. Proc. Natl. Acad. Sci. U.S.A. 100 6263–6268. 10.1073/pnas.0937399100 PubMed DOI PMC

Zadoks J. C., Chang T. T., Konzak C. F. (1974). A decimal code for the growth stages of cereals. Weed Res. 14 415–421. 10.1111/j.1365-3180.1974.tb01084.x DOI

Zong Y., Wang Y., Li C., Zhang R., Chen K., Ran Y., et al. (2017). Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35 438–440. 10.1038/nbt.3811 PubMed DOI

Identification of a Dominant Chlorosis Phenotype Through a Forward Screen of the Triticum turgidum cv. Kronos TILLING Population

See more in PubMed

Dryad
10.5061/dryad.g3r3hp7