Molecular Plant Breeding 2016, Vol.7, No.30, 1
-
6
4
primer set (F: 5-TGC TAC AAG CGT GGA GTG GA-3 and R :5-ACC AGTACA AGG ACG CTT GG-3) and
sequencing of the product (Fitzgerald et al., 2011). The erstwhile mentioned SNP at the splicing donor site of
intron 1 is due to substitution of G by T (Wx
b
allele- low amylose)and it can be identified by allele specific primer
pair F: 5
′
- CCATTCCTTCAGTTCTTTGTCT-3
′
and R: 5
′
-CACTGACCTGGCAAAGAAGG-3
′
). The
primer pair can amplify the fragment containing the first exon
–
intron junction of the Waxy gene (Hoai et al.,
2014).
Recently, several SNP alleles at regulatory region (1514 G/T promoter) and functional regions (T/G intron 1, 1801
T/C exon 9, 2282 A/G intron 10 and 2806 C/T intron 12) of the GBSS I locus have been identified (Biselli et al.,
2014). In this context, dCAPS (derived cleaved amplified polymorphic sequence-Yamanaka et al
.
2004) primers
have been designed to detect SNP for T/G polymorphism (Rathinasabapathi et al., 2015) in intron 1 which explain
77.5% of the total variation in amylose content (Biselli et al., 2014). The dCAPS technique introduces or destroys
restriction enzyme recognition sites by using primers that contain one or more mismatches to the template DNA.
The PCR product modified in this manner is then subjected to restriction enzyme digestion and the presence or
absence of the SNP is determined by the resulting restriction pattern. All primers are designed using the Primer3
0.4.0 software (
/ webcite) and blasted against the rice genomic sequence on the Gramene
website (
to ensure the specificity for the GBSSI gene. In this context, the G-to-T
substitution at the 5
’
leader intron splice donor site of the Wx alleles can be detected by using the primer pair
F:5
’
TGTTGTTCATCAGGAAGAACATCTCCAAG-3
’
and R: 5
’
-TTAATTTCCAGCCCAACACC-3
’
which
generate a unique EcoT14I restriction site characteristic of the Wxa allele. Besides, RM190 (CTn) is identified as
the closely linked microsatellite marker to GBSSI which can explain more than 80% AAC variation (Dobo et
al.,2010). The RM 190 CT repeat primer was designed using the M13-tailed forward primer RM-190 F
(CACGACGTTGTAAAACGA CCTTTGTCTATCTCAAGACAC) and the reverse primer RM-190R
(TTGCAGATGTT CTTCCTGATG) (Chen et al., 2008). Three CT allele variants e.g., CT9, CT10, CT14are
associated with 23
–
24.85% AAC (Tan and Zhang, 2001), while CT11 and CT20 identified accessions with AAC
higher than 25%(Biselli et al., 2014). However, (Temnykh et al., 2000) used the primer pair RM-190F (5
’
-CTTTGTCTATCTCAAGACAC-3
’
) and RM-190R (5
’
-TTGCAGATGTTCTTCCTGATG-3
’
) for genotyping
the RM190 microsatellite CTn alleles.(Fitzgerald et al., 2011) identified three SNPs on the exon 1, 4 and 6 at the
Waxy locus. The SNP for G/T at exon 1 was determined by amplifying a region by primer pair RM190 followed
by a restriction enzyme
“
Acc1
”
(Ayres et al., 1997). Similarly, the G/T polymorphism in intron 1 of the Waxy
gene (In1G and In1T alleles) was genotyped by restriction digest of the PCR fragment of the region amplified
by forward primer RM-190F :5
’
-CTTTGTCTATCTCAAGACAC-3
’
) and reverse primer GBSS-W2R :5
’
-TTTCCAGCCCAACACCTTAC-3
’
) (Ayres et al., 1997). The presence of G and T at the site signifies high or
intermediate amylose, and low amylose status respectively. In fact, haplotypes 8G(identified by RM 190 -CT8
allele), 10G and 11G are associated with the high AAC-types; haplotypes 14G, 16G, 17G, 18G, 19G and 20G are
found in the intermediate AAC-types. Whereas, haplotypes 17T, 18T, and 19T are in the low AAC-types (Chen et
al., 2008). Very low amylose varieties are also associated with SNP on exon 4 (A/G) which can be identified by
the primer pair F: 5
′
- TGC TAC AAG CGT GGA GTG GA-3
′
and R: 5
′
-ACC AGT ACA AGG ACG CTT
GG-3
’
). Intermediate and high amylose varieties were genotyped by SNP status at exon 6 (A/C) using
allele-specific primers (5
’
-CCC ATA CTT CAAAGG AAC ATA-3
′
, 5- GGT TGG AAG CAT CAC GAG TT
–
3 and 5
’
- TCT TCA GGTAGC TCG CCA GT
–
3
’
), where a product size of 292 bp indicates C (intermediate
amylose) and products of 200 and 292 bp identify an A (high amylose). Hence, these molecular markers may be
utilized in masker assisted breeding to develop low GI rice varieties.
Several new SNPs now available by re-sequencing of the Waxy gene and its 1kbp upstream regulatory region
(Biselli et al., 2014). A combination of the CT20 allele of RM190 in combination with the A haplotype for exon 6
and the G haplotype for SNP at 1,514 was always associated to an AAC higher than 24.5%, thus providing an
efficient tool for selecting high AAC rice accessions. The combination of two SNPs in the Waxy gene, including a
single G/T polymorphism at the splicing donor site of the first intron and an SNP in exon 6 potent enough to
