MPB-2016v7n30 - page 10

Molecular Plant Breeding 2016, Vol.7, No.30, 1-6
http://mpb.biopublisher.ca
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gene can unravel novel alleles for use in breeding programme. Eco-tilling is found to be useful to explore the
presence of sequence variation and detection of SNPs in the waxy gene (Hoai et al., 2014; Biselli et al., 2014).
Biselli et al., (2014) sequenced the GBSSI gene as well as 1kbp of the upstream putative regulatory region of
twenty-one genotypes representing all the AAC classes in order to mine the genetic variation. The genome of
Swarna (Low GI) was mapped to high GI (85%) Japonica rice variety “Nipponbare” as reference genome
(Butardo et al., 2011). Rice waxy mutants have no detectable AAC owing to presence of a premature termination
codon in the transcriptional product of the waxy gene (Wanchana et al., 2003). Two wild type waxy alleles,Wxa
and Wxb specific to indica and japonica subspecies cause high and low AAC respectively (Dobo et al.,2010).
Expression level of mRNA and accumulation of waxy protein in Wxa cultivars is 10-fold higher than that of Wxb
cultivars (Isshiki et al., 1998). The SNP involving substitution of G to T at the splicing donor site (AGGTATA to
AGTTATA) of the first intron is designated as Wxb allele which decreases expression level of the GBSS gene
resulting low AAC (Hoai et al. 2014). Among different waxy alleles; wx (recessive) allele is reported to be
associated with highest GI (lowest amylose) while, the Wxa (wild) allele reveals the lowest GI (highest amylose).
In fact, the SNPs at intron1 and exon 6 of GBSS1 are able to explain a maximum of 79.5% of AAC variation
(Biselli et al. 2014). It was found that T/G SNP at position 246,‘A’ at position 2,386, and ‘C’ at position 3,378 in
the GBSS I gene, and C/T SNP at position 1,188 in the glucose-6-phosphate translocator (GPT) gene may
contribute to the low GI phenotype in Swarna from India (GI score 60) and Fedearroz (GI score 50) from
Columbia. (Larking and Park,2003)identified presence of Wxop, Wxin, Wxmq, Wxhp and wx alleles in different
rice varieties using sequence data of exons 4, 6, 5, 2 and 10 of GBSS I. An SNP in exon 6(A to C substitution)
identified as Wxin allele in isogenic lines brings about amino acid substitution from serine to tyrosine (Chen et al.
2008) and results shift in amylose content of the grain from high to intermediate levels (Mikami et al., 2008). Rice
varieties with opaque endosperm show very low amylose (<9%) due to less activity of GBSS l than even low
amylose varieties (Mikami et al., 1999; Mikami et al., 2008) identified an SNP in exon 4 (Wxop allele) associated
with the opaque phenotype which results amino acid substitution of aspartate to Glycine. Low amylose content (<
8%) in most of the tropical glutinous rice is associated a 23-bp duplicated sequence in exon 2 which creates a
premature stop codon at 78bp downstream of the repeated sequence (Wanchana et al., 2003). Jeng et al. (2009)
detected the 23bp duplication in exon 2 in all 35 NaN3-induced waxy mutants derived from rice genotype
Tainung 67 following. Further, a deletion mutation in the Wx gene is shown to be fatal to activity of GBSS l
(Mikami et al., 1999) resulting no biosynthesis of amylose as in IRIS 6-59997(Fitzgerald et al., 2011). Besides,
the range of AAC within each of the erstwhile mentioned five classes suggest the influence of other genes or
genetic background. Thus, understanding the genetic basis of high amylose content in candidate rice varieties will
help in developing low GI rice genotypes to combat diabetic problems.
3 Identification of Molecular Markers
Different allelic variations in the major candidate genes related to starch biosynthesis paves the way for
development of potential molecular markers linked to different classes of amylose content. The association
between AAC and single-nucleotide polymorphisms (SNPs) in the rice Wx gene has been detected at the splicing
donor sites of the intron 1 (Isshiki et al., 1998), exon 4 (Mikami et al., 2008), exon 6 (Larkin and Park, 2003;
Mikami et al.,2008), and exon 10 (Cai et al., 1998; Mikami et al., 2008). The SNPs identified on fourth, sixth
and tenth exon of the GBSS I gene are at 2016(A to C substitution), 2385 (A to C substitution) and 3377(C to T
substitution) position from ATG starting site which resulted non-synonymous changes from Asp to Gly, from Tyr
to Ser, and from Pro to Ser, respectively (Hoai et al. 2014). Similarly, another SNP linked to amylose content
comprised C to T substitution at 3013 position on nineth exon which revealed synonymous change in amino acid.
Cultivars with low amylose had the C SNP in exon 10, whereas all the cultivars with inter mediateand high
amylose was reported to have the T SNP in exon 10(Hoai et al. 2014).The primer pairs designed to identify the
above SNPs onm exon 4-10 are F:5′-TAGCCGAGTTGGTCAAAGGA-3′, R:5′-AAGCACAGGCTGGAGAAAT-3′
and F:5′-TCGCATTGGATGGATGTGTA3 ’, R:5′-GCATAAAACAAAAATGGCATGG-3′ (Hoai et al., 2014).
Besides, very low amylose varieties could be determined by genotyping the SNP on exon 4 (A/G) using the