Grain improvement and Biotechnology toolkit (Module 2)
Overview on aims and the objectives in the 5th project year (June 2009 - May 2010)
The development of nutritionally enhanced types of wheat depends on the availability of variation in grain composition and of tools to select for this variation in plant breeding programmes. Module 2 therefore aims to provide sources of variation of which are appropriate for use by EU plant breeders, and a suite of tools suitable for use in commercial breeding programmes. The objectives for the reporting period included:
- Complete analysis of the HG diversity screen (for dietary fiber components) and to prepare the results for publication.
- Cross triple mutant Sgp-1 (high amylose) lines with 5 commercial varieties to develop lines suitable for use by EU plant breeders.
- Screen the Cadenza mutant population for additional mutations in genes conferring the high amylose phenotype and develop triple mutant lines suitable for introgression into European germplasm.
- Complete characterisation of the transgenic CslF6 (glucan synthase) lines and develop additional transgenic lines to functionally characterise putative xylan synthase and arabinosyl transferase genes.
- Complete proteomic analyses on grain aleurone and starchy endosperm and on genetic stocks to map components to chromosome arms.
- Complete association genetic analysis of WE-AX.
- Carry out genetic analysis of tocol content.
- Establish SNP markers for selected bioactive components.
- Evaluate antibody and inactivated enzyme approaches to develop biochemical kits to quantify arabinoxylans.
- Validate NIR calibrations for arabinoxylans and explore opportunities for exploitation with breeders and NIR instrument manufacturers.
Work performed and the main achievements
WP 2.1 Identification of sources of natural variation in bioactive compounds
The projected targets have been achieved or exceeded in all aspects of the work. The second stage of the diversity screen, to study effects of genotype and environment on a subset of 26 wheat and five rye lines has been completed and the results accepted for publication. This study showed significant differences between groups of bioactive components in the extent to which they varied between samples grown in six different environments, and in the extent to which this variation was heritable. In particular, differences in the contents of sterols, tocols, alkylresorcinols and flour arabinoxylan were highly heritable, while differences in the contents of phenolic acids and folates showed low heritability. Comparisons of methods for analysis of total dietary fibre showed that the values from a novel “difference” method were highly correlated with those obtained using the widely accepted Uppsala method (r2=0.960 for wheat and rye samples, =0.707 for wheat samples only), but were generally lower in absolute terms. Analyses of fractions in the 133 winter wheats showed values ranging from 0.8 to 0.19% dry matter.
A second series of 10 papers has been accepted for publication in Journal of Agricultural and Food Chemistry and the “Healthhgrain Methods Book” published by AACC.
WP 2.2 Generation of new variation in bioactive compounds
Mutagenised populations of bread wheat cultivars Renan and Cadenza have been screened to identify novel mutations in genes controlling key stages in the synthesis of ferulic acid and starch, respectively. Sixteen mutations were identified in the COMT gene controlling ferulic acid synthesis with 3, 8 and 5 mutations on the A, B and D genomes, respectively. These mutations are being combined by crossing to determine effects of the triple mutants on the cross-linking of arabinoxylan. Similarly, 30 mutations in genes encoding starch branching enzyme IIa have been identified and have been crossed to determine their ability to confer a high amylose (resistant starch) phenotype. A second route to resistant starch is also progressing well with a triple mutant line for the Sgp1 (starch synthase II) gene having been crossed with five European cultivars to select lines with 40% amylose (compared with about 25% in commercial cultvars) which are appropriate for use in plant breeding programmes. Transgenesis has been used to confirm the identity of the CslF6 gene as a β-glucan synthase and to provide preliminary characterisation of putative genes encoding xylan sythase and arabinosyl transferase. This provides a basis for identifying and exploiting mutant alleles, as discussed above for ferulic acid synthesis and high amylose starch.
WP 2.3 Development of modern techniques for grain improvement
Following completion of the proteomic map of wheat, work has focused on two topics, assignment of proteins to specific tissues (aleurone and starchy endosperm) and to specific chromosome segments. Genome-wide association genetics has been used to identify seven chromosome regions which are related to arabinoxylan traits, including a major locus on chromosome 6B which is now being characterised by map-based cloning.
An F2 population was analyses to determine the genetic control of tocol content, indicating that two genes may be involved.
The sequences of a number of candidate genes for synthesis of bioactive components (phenolics, arabinoxylan, folate) were determined in 46 wheat lines to identify single nucleotide polymorphisms (SNPs) as markers for variation in content. This showed significant (but unexpected) effects of SNPs in candidate genes for folate synthesis on trienol and stanol content, and of enzymes of lignin biosynthesis and grain phenolic content (phenolic acid and alkylresorcinols).
WP 2.4 The “biotechnology toolkit”
Two complementary approaches have been used to develop bioprobes to quantify feruloylated arabinoxylan, based on monoclonal antibodies and inactivated enzymes. The former has led to the development of a prototype ELISA assay. However, this requires refining for widespread application and talks have been initiated with an SME kit manufacturer to do this. NIR calibration models have been developed for arabinoxylan and arabinoxylan fractions and are being evaluated in collaboration with NIR equipment manufacturers and plant breeders. These three approaches, SNP markers, biochemical kits and NIR calibration, form the basis for the “toolkit” envisaged in the HEALTHGRAIN project proposal.
The development of nutritionally-enhanced wheat varieties depends on the
availability of variation in the amounts
and compositions of key components in wheat and related species, and the
ability of plant breeders to select for
this. Module 2 aims to facilitate improvement by providing such sources
of variation and tools to facilitate their
exploitation.
The objectives for the reporting period were:
WP 2.1 Identification of sources of natural variation in bioactive compounds
The results of the first diversity screen, in which dietary fibre and phytochemical components were determined in 150 wheat lines, 10 rye lines and 30 other cereal lines grown in Martonvásár in 2005, were published in a special section of 11 papers in J. Ag. Food Chem. in November 2008. Based on this study, 26 wheat and five rye lines were selected and grown at Martonvásár for two further years (2006, 2007) and on three additional sites (in Poland, France and the UK) in 2007 only. Grain were analysed for the same groups of bioactive components as in the diversity screen, and will be submitted in a similar series of papers late in 2010. The methods developed and applied in the project have also formed the basis for the “HEALTHGRAIN Methods Book”, to be published by the AACC in autumn 2010.
The data from the six site x year combinations have also been used to calculate the heritability of the different groups of bioactive components: this was particularly high for arabinoxylans and alkylresorcinols but low for folates and phenolic acids.
WP 2.2 Generation of new variation in bioactive compounds
Crosses were performed between the breeding line N11 with mutant forms of the three granule-bound starch synthase II genes (Sgp-A1, -B1, D1) and five modern wheat varieties ( Solstice, Lona, Koreli, Ukrainka and Yumai- 34). The F1 plants were backcrossed once with the same modern varieties using marker-assisted selection to identify triple mutants in the new backgrounds and will be subjected to further rounds of backcrossing to achieve introgression of the mutant genes. The mutations result in a high amylose (resistant starch) phenotype which will be made available to wheat breeders.
A mutant population developed in cv. Cadenza has been screened by TILLING to identify a total of 60 mutations at the three genes encoding starch branching enzyme IIA (wSBEIIa-A, -B and -C). Triple mutants combining null mutations in these three genes should also produce high amylose starch. The new mutations will therefore initially be combined in a common background and then transferred into commercial backgrounds as for the mutant Sgp-1 genes.
Detailed analyses of transgenic wheat lines in which expression of the CSLF6 gene has been down-regulated by RNAi technology have confirmed that it encodes a ß-glucan synthase. Analysis of wholemeal flours showed that the amount of total ß-glucan and the mean molecular weight of the hot water-extractable fraction were both reduced to about half in the RNAi lines compared with those in the wild type plants. Analysis of a second series of lines over-expressing the CSLF6 gene are currently in progress. Similarly, transgenic lines have been produced to HEALTHGRAIN FOOD-CT-2005-514008 12 (178)
over-express and suppress putative xylan synthase and arabinosyl transferase genes. These will be subjected to similar analyses in order to confirm the gene functions.
WP 2.3 Development of modern technologies for grain improvement
Metabolomic analyses have shown that levels of bioactive components in flours may correlate with the concentrations of low molecular mass components in methanol/water extracts analysed by NMR spectrometry, indicating the potential to use this method in high throughput screening.
Detailed proteomic studies of total fractions from aleurone layers and of albumin + globulin fractions from endosperm have been completed. Mass spectrometry was used to identify selected spots including over 400 out of a total of over 1200 spots in the albumin + globulin fraction. This allowed them to be classified into functional groups. Analysis of chromosome deletion lines has also allowed many of the individual components to be assigned to chromosome arms.
Classical genetics has been used to fine map a QTL for content of water-extractable arabinoxylans within an interval of 4cm. Further mapping of regions on seven chromosomes which are associated with the amount and properties of arabinoxylan has been carried out by association genetics, using analytical data for the 150 lines in the diversity screen and DaRT markers for genome mapping. In contrast, a similar analysis failed to show associations between folate content and 12 genes encoding enzymes of folate metabolism. This is consistent with the analysis described in WP2.1 which showed that the folate content has low heritability.
WP 2.4 The “biotechnology toolkit”
Analysis of the lines grown in the diversity screen has allowed the development of robust NIR calibrations for arabinoxylan content. These calibrations may be appropriate for analysis of grain and flour samples in plant breeding and grain trading and links with NIR companies are being developed to evaluate their commercial potential.
New work was commissioned with two HEALTHGRAIN partners to develop biochemical kits to determine arabinoxylan content and properties by exploiting the specific molecular recognition provided by antibodies and inactivated enzymes. The allocation of additional funds for these topics followed our inability to attract new SME partners.
The plant breeding module aims to develop cultivars with increased
contents of bioactive components essential for the delivery of
nutritional benefits to consumers and furthermore to increase these
components in both the endosperm, which is most widely used in food
production, and in the whole grain.
The development of nutritionally-enhanced wheat varieties depends on the
availability of variation in the amounts and compositions of key
components (name the key components in wheat) in wheat and related
species.
Module 2 aims to facilitate improvement by providing such sources of
variation and tools to facilitate their exploitation.
The objectives for the third project year were:
- analyse 26 wheat and five rye lines grown on four sites
- to use transgenesis and mutagenesis to extend the range of variation in the content and composition of dietary fibre and starch
- to establish genomic tools and other "high tech" approaches to facilitate the exploitation of variation
- to develop NIR calibrations for bioactive components.
The work in Module 2 is organised in 4 work packages (WP):
WP 2.1
Identification of sources of natural variation in bioactive compounds
The third field experiment was completed in summer 2007, providing the
final set of material for the diversity screen. This comprised samples
of 26 wheat and five rye lines grown on four sites (in the UK, France,
Hungary and Poland). The same series of analyses were carried out as on
the material from the first two field experiments, for phytochemicals
(sterols, tocols, folates, phenolics acids, alkylresorcinols) and fibre
components (Arabinoxylans, β-glucan).
In addition, betaine and choline were analysed in selected samples.
However, the results obtained so far show clear effects of both genotype
and environment on all components except betaine and choline in which
the main factor of variation is clearly environment.
The distributions of arabinoxylans and phenolic acids within the grain
were determined using novel special imaging methods. These showed
differences in spatial distribution within the grain which could be
exploited by milling.
WP 2.2
Generation of new variation in bioactive compounds
Mutations in genes which are involved in the synthesis of amylopectin
may lead to an increase in the proportion of amylose ("resistant
starch").
Mutagenesis can be used to create novel sources of variation for
exploitation in breeding. Therefore the breadmaking wheat cultivar
Cadenza has been developed, after treatment of seeds with a chemical
mutagen and in the glasshouse and field. These new mutations will be
combined to generate additional mutants while further analyses will
focus on identifying mutations in other genes which will lead to high
amylose starch.
Transgenesis is also being used to confirm the functions of genes
thought to be involved in the biosynthesis of dietary fibre.
WP 2.3
Development of modern technologies for grain improvement
A rapid method for determining choline and betaine in aqueous extracts
of flour, bran and wholemeal has been developed using 1H NMR
spectroscopy. The values for betaine determined by this method have also
been validated by comparison with analyses of the same series of
samples using a classical HPLC-based approach.
The proteomic map of the albumin and globulin fraction from mature grain
of the cultivar Chinese Spring is now essentially complete, with over
350 components having been identified by mass spectrometry. Selected
components are currently being mapped to chromosomes by the analysis of
deletion lines. Similarly over 300 proteins from the aleurone layer have
been analysed by mass spectrometry.
A new strategy has been developed to identify loci and markers for
soluble fibre, exploiting expression profiling and information from the
related regions of the rice genome. The identification of new SNP
(single nucleotide polymorphism) markers for dietary fibre has focused
on an epimerase gene which encodes an enzyme involved in arabinoxylan
synthesis. Sequence analysis of this gene from a number of cultivars has
led to the identification of over 50 mutations in their sequences.
WP 2.4
The "Biotechnology Toolkit"
The availability of detailed analyses of lines from the diversity screen
(WP 2.1) provides an ideal opportunity to develop new analytical
approaches and to evaluate currently available methods for their
applicability to plant breeding programmes. In particular, NIR has been
used to develop calibration for arabinoxylans and other components which
are currently being validated using material from the third field
experiment.
Overview on aims and the objectives for the reporting period
The development of nutritionally-enhanced wheat varieties depends on the
availability of variation in the amounts
and compositions of key components in wheat and related species, and the
ability of plant breeders to select for
this. Module 2 aims to facilitate improvement by providing such sources
of variation and tools to facilitate their
exploitation.
The objectives for the reporting period were:
- to select approximately 25 wheat and 5 rye varieties for detailed analysis
- To use transgenesis and mutagenesis to extend the range of variation in these components
- To establish genomic tools and other “high tech” approaches to facilitate the exploitation of variation
- To develop NIR calibrations for bioactive components and evaluate the use of commercially available kits for fibre analysis
The work in Module 2 is organised in 4 work packages (WP):
WP 2.1
Identification of sources of natural variation in bioactive compounds
A database has been compiled of the amounts and compositions of a range
of phytochemicals (tocols, sterols,
folates, phenolics acids, alkylresorcinols) and fibre components in
approximately 150 wheat lines and 50 other
cereals grown at Martonvasar (Hungary) in 2004-05. Significant variation
in composition was found. For
example, the concentrations of total tocols, total folates and total
alkylresorcinols all varied by more than two-fold
in the 130 winter wheats and of total phenolics acids by more than
three-fold. A similar two-fold variation in total
and water-extractable arabinoxylans fractions was also observed. This
facilitated the selection of 26 wheat and 5
rye lines for future studies, based on differences in composition and
their use as specific genetic stocks. The same
range of analyses has also been carried out on the 31 selected lines
grown at Martonvasar in 2005-06 and they have
been planted in field trials on four sites in different EU member states
(Hungary, France, Poland, UK) for harvest in
2007.
The database was also used to select 50 lines in which the distributions
of water-extractable and waterunextractable
arabinoxylans in the mature grain were mapped using Fourier-transform
infra-red microspectroscopy.
WP 2.2
Generation of new variation in bioactive compounds
Mutagenesis can be used to generate novel sources of variation for
exploitation in breeding. A population of 4,344
lines of the breadmaking wheat cultivar Cadenza has therefore been
developed, after treatment of seeds with the
chemical mutagen ethyl methane sulphonate. Preliminary screening of the
M3 generation grown in the field at
Martonvasar identified a range of phenotypic differences, including
variation in height and flowering time.
The Sgp-1 genes encode a form of granule-bound starch synthase involved
in the synthesis of amylopectin.
Mutations in these genes may therefore lead to an increase in the
proportion of amylose (“resistant starch”),
although it is necessary to combine mutations on all three genomes to
have a significant impact on the amylose
content of hexaploid bread wheat. Natural mutations in the Sgp-A1,
Sgp-B1 and Sgp-D1 loci have therefore been
crossed to generate a triple mutant line. This will now be crossed with
five EU bread wheats to generate high
amylose lines suitable for use in breeding programmes. Analyses of 500
lines of the mutant Cadenza population
discussed above have also identified 13 lines which are null for one of
the Sgp-1 loci.
Transgenesis is also being used to confirm the functions of genes
thought to be involved in the biosynthesis of ß-
glucans and arabinoxylans (dietary fibre). Fourteen constructs have been
made with 11 genes encoding putative
glucan synthases, xylan synthases, arabinoxyl transferases and feruolyl
transferases, in order to determine their
functions by RNAi inhibition and/or over-expression.
This work will identify key genes which can then be targeted to identify
variation in natural and mutant
populations.
WP 2.3
Development of modern technologies for grain improvement
Progress has been made in compiling a proteomic map of mature grain of
the cultivar Chinese Spring, and
assigning the individual components to tissues. 500 proteins from the
aleurone layer have been analysed by mass
spectrometry allowing over 60% of them to be identified and assigned to
functional groups. Similar studies are
now in progress on 600 proteins from the starchy endosperm, following
the development of improved methodology
for protein separation.
A new strategy has been developed to identify loci and markers for
soluble fibre, exploiting expression profiling
and information from the related regions of the rice genome. The
identification of new SNP (single nucleotide
polymorphism) markers for dietary fibre has focused on an epimerase gene
which encodes an enzyme involved in
arabinoxylan synthesis. Sequence analysis of this gene from a number of
cultivars has led to the identification of
over 50 mutations in their sequences.
The development of highly efficient wheat transformation systems has
been completed and these are being
exploited for the functional characterisation of candidate genes in
WP2.2.
WP 2.4
The "Biotechnology Toolkit"
The availability of detailed analyses of 200 cereal lines (WP2.1)
provides an ideal opportunity to develop new
analytical approaches and to evaluate currently available methods for
their applicability to plant breeding
programmes. Evaluation of the total dietary fibre (TDF) kit from
Megazyme showed slightly lower values than
those determined by direct analysis (weight difference). There is also a
need to develop higher throughput
procedures if the method is to be used in plant breeding.
The wheat and rye samples have also been used for NIR analysis, using
wholemeal, white flour and bran fractions.
Preliminary studies have focused on developing calibrations for dietary
fibre and alkylresorcinols.
The further development of work in this module requires the input of new
partners specialising in the commercial
development of analytical tools and kits. Up to three SME partners with
relevant expertise will therefore be sought
in the call for new partners to be published in July 2007.
Module 2 aims to provide nutritionally-enhanced varieties of cereal (wheat and rye) as improved raw material for
the food processing industry. The module has two major parts: the identification and generation of new variation
in grain composition and the development of methods (both high-tech and simplified) to facilitate the
incorporation of this variation into cultivars suitable for production in the EU.
The objectives for the first year were:
- To determine the range of diversity in the content of bioactive compounds in a collection of wheats and other cereals selected for their wide genetic diversity
- To initiate work on increasing the range of variation in composition in EU wheats by introgression of genes from exotic lines, chemical mutagenesis and transgenesis
- To develop a suite of underpinning technologies (genomics, genetics, metabolomics, proteomics) to facilitate the exploitation of variation and to form the basis for the development of a simplified “tool kit” for plant breeders
Work performed and the main achievements in the 1st project year (June 2005 - May 2006)
