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: rita.armoniene@lammc.lt

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.