Download Identification of Novel Starch Traits in Sorghum

IAEA-CN-167-011P
Identification of Novel Starch Traits in Sorghum
(Sorghum bicolor. L) : A Reverse Genetics Approach
Helen Hill, L.Slade Lee and Robert Henry
Grain Foods CRC Limited, Centre for Plant Conservation Genetics, Southern Cross University P.O. Box 157, Military Road Lismore 2480 Australia.
Introduction
Methods
Mature seeds of Sorghum bicolor L (cv. MR 43)
were bombarded with gamma radiation to induce
mutation. Following treatment, over 1000 irradiated
seeds were grown in a glasshouse at 25-30°C with
16 hours day length. M2 leaf samples were collected
and genomic DNA was extracted utilising a Qiagen
96-well MagAttract kit on the MWG Theonyx® robotic
platform. PCR was performed to amplify our genes
of interest and then directly sequenced. Following
sequencing all individual data was manually visualised
and aligned using SEQUENCHER™ 4.0 Software (Gene
Codes Corporation, USA) and compared to published
sequence in Genbank.
Sorghum (Sorghum bicolor L.) is the fifth most important cereal grain crop
in the world and reportedly feeds over 500 million people on a daily basis
in the developing world providing dietary starch, dietary protein and some
vitamins and minerals. Traditionally, sorghum grain is used for thin and
thick porridges, noodles, tortillas, flatbread, biscuits, snacks, couscous,
and traditional beer. In the West, it is predominantly used as an animal
feed and is becoming important for ethanol production. More recently,
specialised sorghum starch has been used for food and industrial uses.
The aim of this study is to use a reverse genetics approach to detect
DNA base changes in four important starch synthesis genes encoding;
soluble starch synthase (SS I and SS IIa), granule bound starch synthase
(GBSS) and starch branching enzyme (SBE IIb) in our gamma irradiated
sorghum (cv. MR 43) population and to correlate sequence variation to
altered starch phenotype which may subsequently by used for novel food
applications.
GBSS
SS IIa
Results
We have surveyed over 3.3 kb of the GBSS gene, 2.5 kb region of SBE IIb, 1kb region
of SS IIa gene and 400bp of the SS I gene and found numerous single nucleotide
polymorphisms (SNP) and insertion/deletions (indels) in intronic regions and also some
single nucleotide polymorphisms in exonic regions of the genes of interest. (Table 1)
Table 1 summarises the natural variation found between the putative sorghum mutants and
the published Genbank sequence for GBSS, SS IIa, SS I and SBE IIb respectively, which
consisted of indels and base substitutions.
Gene product
Abbreviation
Granule bound
starch synthase
GBSS
Starch branching
SBE II
enzyme
Figure 1 shows the difference in growth of the sorghum
seedlings, after the seed was subjected to different
gamma radiation doses.
Conclusions
Soluble starch
synthase
Soluble starch
synthase
Length
Summary of findings
studied
13 SNPs; including transitions being CgT, AgG
or GgA base change and transversions being a
3.3kb
AgC, TgA, TgG and GgT and a single dinucleotide
AAgTC snp ; and 4 indels including an AT , T , TT
and TATGTAGT indel
8 SNPs; including transitions AgG, TgC and
GgA; transversions being TgA, TgG, CgG and a
2.5kb
dinucleotide snp TTgAG; and 4 indels including an
AA, ACG, AT and ATA indel.
SS IIa
1kb
1 SNP being CgT change in exon
SS1
400bp
1 SNP being GCgCA in exon
Whereas mutations, in the form of single nucleotide
polymorphisms (SNP) and insertion/deletion (indel)
events, predominantly occurred in non-coding
introns, some mutations were also found in exons
in all genes studied. These SNP changes resulted in
some differences in the amino acids in each of the
starch synthesis genes of interest and which leads to
alternative proteins encoded and may subsequently
result in an altered starch phenotype in our mutant
sorghum.
Phenotypic analyses will be carried out to correlate sequence variation to altered starch
phenotype. Screening of diverse sorghum germplasm by the EMAIL technique developed at
CPCG is currently proceeding.
SCU2554
Further Work