RESUMEN
BACKGROUND: Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes. RESULTS: Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry. CONCLUSIONS: We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/genética , Amilosa/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Genoma de Planta/genética , Polimorfismo de Nucleótido Simple/genética , Triticum/genética , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Alelos , Sustitución de Aminoácidos , Amilosa/análisis , Amilosa/genética , Cruzamiento , ADN de Plantas/genética , Endospermo/química , Endospermo/enzimología , Endospermo/genética , Genotipo , Datos de Secuencia Molecular , Mutación , Fenotipo , Hojas de la Planta/química , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poliploidía , Isoformas de Proteínas/genética , ARN de Planta/genética , Análisis de Secuencia de ADN , Almidón/química , Almidón/genética , Almidón/metabolismo , Triticum/química , Triticum/enzimologíaRESUMEN
Glyphosate (N-phosphonomethyl-glycine) is the world's most widely used broad spectrum, post-emergence herbicide. It inhibits the chloroplast-targeted enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), a component of the plant and microorganism-specific shikimate pathway and a key catalyst in the production of aromatic amino acids. Variants of EPSPS that are not inhibited by glyphosate due to particular amino acid alterations in the active site of the enzyme are known. Some of these variants have been identified in weed species that have developed resistance to glyphosate because of the strong selective pressure of continuous, heavy glyphosate use. We have used TILLING (Targeting Induced Local Lesions in Genomes), a non-transgenic, target-selected, reverse genetics, mutation breeding technique, and conventional genetic crosses, to identify and combine, through two rounds of mutagenesis, wheat lines having both T102I and P106S (so-called TIPS enzyme) mutations in both the A and the D sub-genome homoeologous copies of the wheat EPSPS gene. The combined effects of the T102I and P106S mutations are known from previous work in multiple species to minimize the binding of the herbicide while maintaining the affinity of the catalytic site for its native substrates. These novel wheat lines exhibit substantial tolerance to commercially relevant levels of glyphosate.
Asunto(s)
Glicina/análogos & derivados , Triticum , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Mutación , GlifosatoRESUMEN
We report the use of TILLING (targeting induced local lesions in genomes), a reverse genetic, nontransgenic method, to improve a quality trait in a polyploid crop plant. Waxy starches, composed mostly of amylopectin, have unique physiochemical properties. Wheat with only one or two functional waxy genes (granule-bound starch synthase I, or GBSSI) produces starch with intermediate levels of amylopectin. We have identified 246 alleles of the waxy genes by TILLING each homoeolog in 1,920 allohexaploid and allotetraploid wheat individuals. These alleles encode waxy enzymes ranging in activity from near wild type to null, and they represent more genetic diversity than had been described in the preceding 25 years. A line of bread wheat containing homozygous mutations in two waxy homoeologs created through TILLING and a preexisting deletion of the third waxy homoeolog displays a near-null waxy phenotype. This approach to creating and identifying genetic variation shows potential as a tool for crop improvement.
Asunto(s)
Plantas Modificadas Genéticamente , Transgenes , Triticum/genética , Alelos , Amilopectina/metabolismo , Amilosa/química , Productos Agrícolas/genética , Análisis Mutacional de ADN , Cartilla de ADN/química , Eliminación de Gen , Biblioteca de Genes , Genes de Plantas , Genoma , Genoma de Planta , Heterocigoto , Homocigoto , Modelos Genéticos , Mutación , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
Transgenic methods have been successfully applied to trait improvement in a number of crops. However, reverse genetics studies by transgenic means are not practical in many commercially important crops, hampering investigations into gene function and the development of novel and improved cultivars. A nontransgenic method for reverse genetics called Targeting Induced Local Lesions IN Genomes (TILLING) has been developed as a method for inducing and identifying novel genetic variation, and has been demonstrated in the model plant, Arabidopsis thaliana. Recently, TILLING has been extended to the improvement of crop plants and shows great promise as a general method for both functional genomics and modulation of key traits in diverse crops.