Table 2.
The recent achievements of TILLING in basic studies of gene function and/or breeding of commercial cultivars.
| Category | Goal | Species/cultivar | Gene(s) | Function | Investigated mutant(s) | Effect of mutation | References |
|---|---|---|---|---|---|---|---|
| Increasing drought tolerance | Barley (H. vulgare), cv. Sebastian | HvABI5 | ABA Insensitive 5, a basic leucine zipper (bZIP) transcription factor | hvabi5.d carrying a missense mutation | Increase tolerance to drought stress by better membrane protection, higher flavonoid content, and faster stomatal closure | Collin et al., 2020 | |
| HvCBP20 | Cap-Binding Protein 20 - small subunit of nuclear Cap-Binding Complex (nCBC) | hvcbp20.ab carrying a missense mutation | Increase tolerance to drought stress by lower permeability of epidermis (increased wax deposition) | Daszkowska-Golec et al., 2020 | |||
| HvERA1 | Enhanced Response to ABA1, β-subunit of farnesyltransferase | hvera1.b carrying a missense mutation | Increase tolerance to drought stress by better photosynthesis performance and a higher leaf RWC; semi-dwarf phenotype and ABA-sensitivity during seed germination | Daszkowska-Golec et al., 2018 | |||
| Uncovering the role of strigolactones in response to drought | HvD14 | Strigolactones receptor | hvd14.d carrying missense mutation | Hyper-sensitive to drought stress | Marzec et al., 2020 | ||
| Increasing plant tolerance to salinity |
Yellow mustard (Brassica rapassp. trilocularis), Inbred line R-o-18 s |
CAX1a | Ca2+ cation exchangers (CAX) transporter | BraA.cax1a-7, BraA.cax1a-4, BraA.cax1a-12 carrying missense mutations |
BraA.cax1a-4 provided higher biomass, better photosynthetic performance - higher water use efficiency, Fv/Fm, electron fluxes, and Rubisco values; mutants presented increased osmotic protection through a myo-inositol accumulation |
Navarro-León et al., 2021 | |
| Decreasing heavy metal accumulation | Tobacco (N. tabacum), Yunyan 87 |
HMA2S HMA4T | Heavy metal transporters (Cd and Zn) |
Nonsense (hma2s-7) missense (hma4t-3) |
The levels of Cd and Zn decreased in leaves which was confirmed by CRISPR/Cas9- knockout mutants | Gao et al., 2020 | |
| Alleviating Zn deficiency and toxicity, improvement in Cd phytoremediation programs | Yellow mustard (Brassica rapa ssp. trilocularis), inbred line R-o-18 |
HMA4a | Heavy metal ATPase or Heavy Metal Associated (HMA), transporter which is able to transport Cd or Zn from the root to the shoot | Mutant carrying a missense mutation (BraA.hma4a-3), identified by Lochlainn et al., 2011 |
Higher tolerance to Cd and Zn |
Blasco et al., 2019; Navarro-Leon et al., 2019a |
|
| Increasing aluminium tolerance, functional analysis of ATR in barley | Barley (H. vulgare), cv. Sebastian | HvATR | Ataxia Telangiectasia and Rad-3-related protein kinase, involved in DDR pathway | hvatr.g and hvatr.i mutants carrying a missense mutation | Altered Aluminium response – accumulation of DNA damage; the frequency of cell division in roots not reduced after Al treatment | Szurman-Zubrzycka et al., 2019b | |
| Cold response, adaptation to chilling stress | Rice (O. sativa), japonica, cv.Zhonghua 11 (ZH11) | OsCIPK7 | Calcineurin B-like interacting protein kinases | Three mutants (two with missense, and one with nonsense mutation) | Increased chilling tolerance (one mutant with missense mutation) | Zhang et al., 2019 | |
| Improvement in the thermotolerance of tomato varieties | Tomato (Solanum lycopersicum), cv. Red Setter | HSBP1 | Heat Shock Binding Protein 1 |
One mutant with a missense mutation, identified by Minoia et al., 2010 | Enhanced basal thermotolerance, mature plants exhibit increased resilience under repeated HS treatments | Marko et al., 2019 | |
| Product quality | Improving seed oil composition - elevating oleic acid and lowering polyunsaturated fatty acid contents | Soybean (Glycine max L. Merr.), cv. Forrest |
GmSACPD-C
GmFAD2-1A GmFAD2-1B GmFAD3A-C |
Fatty acid desaturases that control saturated/unsaturated fatty acid ratio | In total missense – 92 nonsense - 5 |
Fatty acid phenotypes of the 24 mutants have been improved (missense, nonsense) | Lakhssassi et al., 2021b |
| Low phytic acid mutants identification | Oilseed rape (Brassica napus), inbred line Express 617 | Bn2-PGK | 2-phosphoglyceric acid kinase involved in the phytic acid synthesis pathway | Four mutants: two carrying mutations in a single gene, two carrying mutations in two paralogs | 2-PGK2 (2-phosphoglyceric acid kinase) double mutants had significantly reduced phytic acid contents |
Sashidhar et al., 2020 | |
| Development of reduced-immunogenicity wheat genotypes relevant to most gluten-sensitive individuals with celiac disease | Common wheat (T. aestivum), cv. Express; Durum wheat (T. durum),cv. Kronos |
DME
DRE2 |
DEMETER, 5-methylcytosine DNA glycosylase/lyase causes demethylation; DRE2 an iron-sulfur cluster biogenesis enzyme | Double mutants in durum wheat and triple mutants in common wheat with complete DME or DRE2 activity suppression |
Mutants displayed reduced content of immunogenic gluten proteins while retaining essential baking properties |
Wen et al., 2022 | |
| Elevating the levels of resistant starch, an important component of dietary fiber, associated with health benefits such as reduced glycaemic response | Common wheat (Triticum aestivum), cv. Cadenza | SSIII | Starch synthase III plays a key role in starch biosynthesis (synthesis of long amylopectin chains) | Triple ssIIIa mutants carrying mutations in each homoeologous copy of ssIIIa (A, B, and D) | Starch chain length distributions, increased levels of amylose, and fewer long amylopectin chains. Mutants, had more resistant starch and greater levels of non-starch polysaccharides | Fahy et al., 2022 | |
| Increasing the resistant starch content (human health benefits) | Common wheat (T. aestivum); cv. Jagger, hard red winter cultivar | SSIIa | Starch Synthase | Triple null mutants | High amylose and resistant starch | Schoen et al., 2021 | |
| Enrichment of provitamin A content in durum wheat grain | Durum wheat (T. durum), cv. Kronos | HYD1 | β-carotene hydroxylase 1 | Mutation in A and B subgenomes (a splice site, nonsense) | Increase of β-carotene by more than 70% | Garcia Molina et al., 2021 | |
| lcyE | Lycopene ϵ-cyclase | Mutation in A and B subgenomes (nonsense, a splice site) | Increase in β-carotene content by roughly 75% | Sestili et al., 2019 | |||
| Development of non-transgenic glyphosate-tolerant wheat | Common wheat (T. aestivum), cv. Express | EPSPS | Enzyme 5-enolpyruvylshiki- mate-3-phosphate synthase | Double mutant with missense mutations in A and D subgenome | Mutant exhibits substantial tolerance to commercially relevant levels of glyphosate | Moehs et al., 2021 | |
| Investigating if CAX1 modifications have effects on plant metabolism |
Yellow mustard (Brassica rapa ssp. trilocularis), inbred line R-o-18 |
CAX1a | Cation/H+ exchanger transporters | Three mutants carrying missense mutation, identified by Lochlainn et al., 2011 | Inhibited some N metabolism enzymes, activated photorespiration activity, increased tolerance to high Ca2+ | Navarro-Leon et al., 2019b | |
| Improvement of fruit shelf-life | Tomato (Solanum lycopersicum), line M82 |
SlACO1
SlE8 |
ACC oxidase 1 | In total 6 mutants (missense) | Mutants slaco1-1 and slaco1-2 showed decreased ethylene production and conductivity, enhanced shelf-life and firmness; sle8-1 showed enhanced ethylene levels and reduced shelf-life, accelerate ripening | Brisou et al., 2021 | |
| Increasing the grain size and weight |
TILLING mutants were identified in durum wheat (T. durum), cv. Kronos. The mutations were introduced to common wheat (T. aestivum), cv. Paragon |
TaGW2 | RING-type E3 ubiquitin ligase takes part in the ubiquitin-proteasome pathway and regulates cell division | Seeds of the Paragon BC1 NIL (Near-isogenic lines) carrying the three TILLING-derived mutant alleles | Mutants carrying single-copy nonsense mutations in different genomes displayed an increase in GS and TGW. The enhanced effect was visible in the double and triple mutants. |
Wang et al., 2018 | |
| Investigating the role of SS4 in wheat | Durum wheat (T. durum), cv. Kronos | SS4 | Glucosyltransferase, Starch Synthase 4 |
Mutation in A and B subgenomes (nonsense, a splice site) | Alterations in endosperm starch granule morphology; during early grain development, most amyloplasts in the mutant formed compound granules due to multiple initiations; reduced starch content in leaves and pollen grains | Hawkins et al., 2021 | |
| Improvement of lignocellulosic quality of sorghum | Sorghum (Sorghum bicolor), line BTx623 |
Bmr19 locus;
FPGS gene |
Enzyme involved in folate (C1) metabolism, putative folylpolyglutamate synthase |
Four mutants (missense), one with leaf Midrib phenotype (brown) | Reductions of the lignin content in the biomass, impair lignin biosynthesis. | Adeyanju et al., 2021 | |
| Development of pea varieties lacking saponins in their seeds. that can impart an undesirable bitter taste | Pea (Pisum sativum), winter pea line Ps336/11 and Caméor spring pea |
PsBAS1 | β-amyrin Synthase 1 |
In Ps336/11 TILLING population: in total 8 mutations, one nonsense; in the Caméor TILLING population: in total 17 mutations: 3 intronic, 3 silent, 10 missense, 1 splice site |
Two homozygous mutants seeds accumulated virtually no saponin, identified in two genetic backgrounds (Ps336/11, Caméor) | Vernoud et al., 2021 | |
| Other traits and processes | Examination of the role of wheat OMT2 in methylated flavone biosynthesis | Durum wheat (Triticum turgidum L), cv. Kronos | OMT2 | O-methyltransferase catalyzes an O-methylation of the hydroxyl groups in flavones | Loss-of-function mutants of OMT2 homoeologs (omt-A2 and omt-B2); double mutant | Increased levels of chlorogenic acid in glumes of a mutant is suggesting that it might serve as a substrate for OMT2 | Cain et al., 2022 |
| Functional analysis of DMC1 in barley involved in DNA repair | Barley (H. vulgare), cv. Sebastian | HvDMC1 | Disrupted Meiotic cDNA1 recombinase that takes part in the repair of double-strand break (DSB) | hvdmc1.c carrying a missense mutation | Increased frequency of all chromosome aberrations during meiosis | Szurman-Zubrzycka et al., 2019a | |
| Generation of semi-dwarf and dwarf genotypes to study the relevance of brassinosteroids in barley development | HvDWARF | BR-6-oxidases, involved in brassinosteroids biosynthesis | Seven missense, one splice site | The short stature of various degrees and disturbance in brassinosteroids biosynthesis (mutation at a splice site led to dwarf phenotype, three missense to semi-dwarf mutants) | Gruszka et al., 2016 | ||
| Checking whether NLP genes in the Triticeae crops are involved in nitrate regulation and nitrogen use efficiency (NUE) | Barley (Hordeum vulgare L.), cv. Tamalpais | HvNLP2 | Nodule Inception-like Protein that plays essential roles in nitrate signaling | hvnlp2-1 carrying a missense mutation | Nitrate content is significantly higher, which may result from the decreased assimilation of nitrogen caused by reduced nitrate reductase activity and expression of nitrate assimilatory genes; mutants exhibited reduced biomass, seed yield, and NUE |
Gao et al., 2022 | |
| Investigating the role of OsERL in rice | Rice (Oryza sativa L.), Zhonghua 11 (ZH11) | OsERL | Leucine-rich repeat receptor-like kinase | In total 76 mutants, 19 missense, and 2 nonsense | Defects in the anther development, male sterility, or reduced numbers of anther lobes (surprisingly, two nonsense mutations led to a moderate effect, and two missense mutations led to a strong effect) | Liu et al., 2021 |
FAD2, delta(12)-fatty-acid desaturase; Ara h 1, vicilin; SACPD, delta-9-stearoyl-acyl carrier protein desaturase catalyzes the conversion of stearic acid to oleic acid; FAD2, the omega-6 fatty acid desaturase 2 converts oleic acid into linoleic acid; FAD3, the microsomal omega-3 fatty acid desaturase converts linoleic acid to linolenic acid.