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

Abstract

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.

Keywords: TILLING; bulked-segregant analysis; chlorosis; durum wheat; genomics; mapping-by-sequencing; senescence.

FIGURE 1

A premature yellowing phenotype from the Kronos TILLING population segregates as a single dominant locus. F₂ populations of the K2282 Kronos TILLING line grown at the JIC in 2016 showed an early yellowing phenotype (A) Pigment content was measured in the yellow mutant plants (F₂M) compared to the wild-type plants (F₂WT) (B; n = 3 per genotype) and was also quantified using SPAD (C; n = 153 F₂M, n = 61 F₂WT). The yellow group (F₂M) senesced significantly earlier than the late bulk (F₂WT) (D; n = 148 F₂M, n = 56 F₂WT). Scoring of the plants demonstrated that the F₂ population was segregating 3:1 for the yellow trait, indicative of a dominant single locus where heterozygous plants, such as the F₁ generation, also present the mutant phenotype (E; numbers are combined for both populations). F₂M and F₂WT refer to plants which are yellow and green, respectively, and which derive from the F₂ population (see bottom of E), while WT and MP refer to Kronos WT plants or M₄ K2282 plants, respectively (see top of E).

FIGURE 2

Bulked segregant analysis identifies the YES-1 locus on chromosome 3A. Exome capture was carried out on yellow and green bulks from K2282 × Kronos F₂ populations grown at the JIC in 2016. The K2282-A yellow bulk (yellow line, inner track; smoothed to a moving average of 4) and green bulk (green line) were scored at each SNP locus identified for enrichment of the mutant allele. The level of enrichment in the green bulk was subtracted from that of the yellow bulk to obtain the Δ value (outer track; smoothed to a moving average of 4). A high Δ value, indicative of a region enriched for mutant alleles within the yellow bulks, was identified on the long arm of chromosome 3A (B; smoothed to a moving average of 4). Markers designed on known TILLING SNPs within this region mapped the YES-1 locus to a 32.9 Mb interval within the F₂ population (C). Green bars indicate wild-type calls, while yellow bars indicate mutant or heterozygous calls. The relevant phenotype of each recombinant group is shown to the left of the panel. The numbers of individual plants that fell into each recombination interval are shown to the right. The chromosome scale in (A) is given in Mb.

FIGURE 3

The YES-1 locus causes lower chlorophyll levels before anthesis and earlier onset of senescence. The early chlorosis phenotype was recapitulated in the JIC 2018 field trials. Pigment content in the mutant lines is significantly lower at the third leaf stage (Zadoks 13–14, 24 days before anthesis) and becomes more extreme by anthesis (A; ^∗∗p < 0.01; ^∗∗∗p < 0.001 Student’s t-test). Relatively chlorophyll content, as measured with a SPAD meter, is significantly decreased in the mutant lines before anthesis, and remains significantly lower until 29 days post-anthesis (B; ^∗∗p < 0.01; ^∗∗∗p < 0.001 Pairwise Wilcoxon Rank Sum, adjusted for FDR). The yellowing phenotype in the leaves were clear in the field at 20 DPA (C).

FIGURE 4

The YES-1 locus fine-maps to a 4.3 Mb region containing 59 genes. Markers were developed for novel SNPs identified in non-coding regions. We used phenotypic data from JIC 2017 and 2018 field trials to classify recombinant lines as green or yellow, indicated to the right of the panel. (A). These markers mapped YES-1 to a 4.3 Mb interval between markers SH044 and SH59985. Expression data for the 59 high-confidence genes in the region (B) from developmental time course data (Ramírez-González et al., 2018) highlights gene expression in root (yellow, top), leaf/shoot (orange, second from top), spike (green, second from bottom) and grain (purple, bottom) tissues across developmental stages. Genes mentioned in the text are highlighted by an asterisk (TraesCS3A02G412900 to TraesCS3A02G413200; OsSAG12 orthologs), a circle (TraesCS3A02G410800; Tryptophan Decarboxylase 2) or an inverted triangle (TraesCS3A02G414000; putative magnesium transporter). The full list of genes is provided in physical order in Supplementary Table 7.