LAMMC
Rita Armonienė and Gintaras Brazauskas
Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry, Instituto a. 1, LT58344 Akademija, Kėdainiai distr., Lithuania. E-mail: [email protected]
INTRODUCTION
Waxy WT M300 M301 M302 M303 M305 M306 M309 M310 M312 M313 M314 M317
M317
Waxy WT M318 M319 M320 M321 M322 M323 M327 M328 M329 M330
Waxy WT M625 M264 M267 M288 M289 M290 M291 M292 M294 M295 M296
FUTURE WORK
CONCLUSIONS
TILLING is a powerful forward or reverse genetic
tool for development of new alleles in genes of
interest. This TILLING population will serve as a
valuable source of novel alleles of starch
biosynthesis genes in order to develop winter
wheat cultivars with various amylose content. • to identify amylose content variation in 756
genotypes of M3 TILLING population using
traditional I2–KI dyeing method;
• to estimate precise amylose and resistant
starch content in selected genotypes;
• to make sequence analysis of key starch
biosynthesis genes of selected genotypes in
order to identify novel alleles;
• to confirm impact of mutations in starch
biosynthesis genes by gene expression
analysis.
TILLING population of winter wheat
Winter wheat
‘Kena DS’ and ‘Gaja DS’
EMS
M0 seeds
M1 generation
M2 generation (DNA sampling)
M2 seeds M3 seeds
Reverse Genetics
Gene of interest Mutation identification
Phenotypic analysis
Forward Genetics
Gene/mutation identification
Screening of mutants with desired phenotype
Biological process of interest
Starch is a main component of wheat grain and consists of two glucan polymers amylose and amylopectin with the ratio ranging
between 20-30 and 70-80%, respectively (Yu et al., 2012). The amylose / amylopectin ratio has a major influence over the
physiochemical properties of starch and determines its optimal application in the industry. The isoforms of granule-bound starch
synthase (GBSS) are responsible for the biosynthesis of amylose, whereas amylopectin synthesis is a complex pathway that
involves at least three starch synthases (SSI, SSII, SSIII) and several branching (SBEI, SBEIIa and SBEIIb) and de-branching
enzymes (DBE) (Yamamori et al., 2006). Recently, amylose-free (Waxy) and high-amylose wheats, consisting up to 100%
apylopectin and 70% amylose, respectively, were produced through the development of new biotechnology techniques. The
starches of these wheat provide the unique starch functional properties that are desirable for food and non-food industries (Sestili et
al., 2014). In this study, Targeting Induced Local Lesions in Genomes (TILLING) population of the two winter wheat cultivars (‘Kena
DS’ and ‘Gaja DS’) were used as source of new alleles in starch biosynyhesis genes responsible for amylose variation in the starch.
Fig.1 The staining technique based on an i2-KI solution.
Wheat grains containing amylose (blue), amylose-free
‘Waxy ‘ (light brown.)
References:
Sestili F., Botticella E.and Lafiandra D.2014. TILLING for Improved
Starch Composition in Wheat R. Tuberosa et al. (eds.), Genomics of
Plant Genetic Resources, 467-487.
Yamamori M, Kato M, Yui M, Kawasaki M.2006. Resistant starch and
starch pasting properties of a starch synthase IIa-deficient wheat with
apparent high amylose. Aust J Agric Res 57:531–535.
Yu CH, Bie TD, Wang C, Zhang X, Wu RL, Cheng XM, Wang CG,
Zhao Y, Cheng SH. 2012. Development of near-isogenic lines with
different wheat Wx genes and their effects on amylose content and
noodle quality. Acta Agron Sin 38:454–461.
I2-KI test of TILLING population
(M3 generation / 746 genotypes)
Amylose and resistant starch
content analysis
(M4 generation / ~100 genotypes)
Sequencing GBSSI, SSII
(M4 generation /
~50 genotypes)
Expression analysis
GBSSI, SSII
(M4 generation /
~ 2-3)
Fig. 2 Study scheme.
Fig. 3 I2–KI staining test of winter wheat population. WT-wild type ‘KenaDS’, Waxy - amylose free
genotype (control). Blue color fraction indicates amylose in the starch.
OBJECTIVES MATERIALS AND METHODS:
• to identify new alleles of starch biosynthesis
genes responsible of amylose content variation
in the starch;
• to implicate identified alleles into the breeding
programs of winter wheat with novel starch
composition.
Two seeds per sample were ground using mortar and pestle and
stained with iodine solution (0.025% I2–KI). Samples were left to
precipitate out at room temperature for 24 hours. Genotypes that
endosperm showed smaller or higher amount of stained amylose
(blue color band) comparing with wild type were chosen for further
analysis.
RESULTS
Three hundred M3 generation genotypes of
TILLING-population and its wild type cultivar
‘Kena DS’ and Waxy wheat breeder’s line as
control were chosen to identify variation for
amylose content using I2–KI solution (Fig.3).
Narrow blue color band indicating low amylose
content was observed in 25 M3 genotypes
while 10 M3 genotypes showed wider blue
color band in comparison with wild type .
Pilot experiment to evaluate amylose content variation in our winter wheat TILLING
population was carried out. Mutation density of one mutation per 37.84 Kb of the
DNA was previously determined in this TILLING population while screening
freezing tolerance associated genes.
