Riccardo Bovina

TILLING mutants of durum wheat result in a high amylose phenotype and provide information on alternative splicing mechanisms.

The amylose/amylopectin ratio has a major influence over the properties of starch and determines its optimal end use. Here, high amylose durum wheat has been bred by combining knock down alleles at the two homoelogous genes encoding starch branching enzyme IIa (SBEIIa-A and SBEIIa-B). The complete silencing of these genes had a number of pleiotropic effects on starch synthesis: it affected the transcriptional activity of SBEIIb, ISA1 (starch debranching enzyme) and all of the genes encoding starch synthases (SSI, SSIIa, SSIII and GBSSI). The starch produced by grain of the double SBEIIa mutants was high in amylose (up to ∼1.95 fold that of the wild type) and contained up to about eight fold more resistant starch. A single nucleotide polymorphism adjacent to the splice site at the end of exon 10 of the G364E mutant copies of both SBEIIa-A and SBEIIa-B resulted in the loss of a conserved exonic splicing silencer element. Its starch was similar to that of the SBEIIa double mutant. G364E SBEIIa pre-mRNA was incorrectly processed, resulting in the formation of alternative, but non-functional splicing products.

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.

Identification of root morphology mutants in barley

In this study, a forward-genetics analysis was performed on a portion of TILLMore, a chemically mutagenized population of barley cv. ‘Morex’ (http://www.distagenomics.unibo.it/TILLMore/), to identify root morphology alterations by comparison with ‘Morex’ wild-type. For this purpose, a simple paper-roll approach was performed to identify phenotypic variants at the seedling stage. The analysis of c. 1000 M4 families allowed us to identify c. 70 lines with altered root morphology. A more accurate phenotypic characterization of a portion of the mutant lines has been performed using stereomicroscopy and a scanning electron microscopy approach.

Starch metabolism mutants in barley: A TILLING approach

In this study, the targetting-induced local lesions in genomes approach was used to identify mutants for genes related to starch metabolism in barley. Starch is the major reserve of plants and serves as primary carbohydrate component in human and livestock diets and has also numerous industrial applications. Mutants for biosynthetic or regulatory genes of starch metabolism often produce starch granules with abnormal morphological and molecular features that could be of interest for technological applications. We report the identification of 29 mutations in five starch-related barley genes ( Bmy1 , GBSSI , LDA1 , SSI and SSII ) through the molecular screening of TILLMore, a sodium azide-mutagenized population. Almost all the mutations detected were CG–TA transitions and several ( c. 60%) implied a change in amino-acid sequence and therefore possible phenotypic effects. Four mutants showed non-sense or splice-junction alterations, which could drastically affect the protein function.